Nitrile hydratase variant

ABSTRACT

A nitrile hydratase variant of the present invention comprises substitution of at least one amino acid with another amino acid to improve two or more properties of nitrile hydratase by substitution of one amino acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.13/128,323, filed May 9, 2011, which is a U.S. National StageApplication of PCT/JP2009/006055, filed Nov. 12, 2009, which claimspriority to Japanese application number 2008-292819, filed Nov. 14,2008, the entire contents of which are incorporated by reference hereinin their entirety.

TECHNICAL FIELD

The present invention relates to a nitrile hydratase variant, a geneencoding the nitrile hydratase variant, a DNA containing the gene, aplasmid containing the gene, a transformant by means of the plasmid, anda method for producing a nitrile hydratase variant using thetransformant.

BACKGROUND ART

In recent years, a nitrile hydratase has been discovered which is anenzyme having the nitrile-hydrating activity to convert a nitrile groupof various compounds to an amide group by hydration, and a number ofmicroorganism strains producing the above-mentioned enzyme have beendisclosed. In order to produce an amide compound from a nitrile compoundusing a nitrile hydratase on an industrial scale, it is important toreduce the production costs for this enzyme in the total productioncosts for producing the amide compound. More specifically, it isnecessary to increase the activity value in a unit weight of thepreparation obtained from the enzyme production.

As a method for increasing the activity value by increasing the amountof the enzyme in the enzyme preparation, attempts have already been madeto clone the gene encoding the above-mentioned enzyme for the purpose ofexpressing a large amount of the enzyme through genetic engineeringmethods.

For example, there are produced a plasmid expressing a large number ofthe Pseudonocardia thermophila-derived nitrile hydratase in thetransformant and a transformant strain transformed with the plasmid. Inaddition, it has been made possible to produce a nitrile hydratase bymeans of these transformant strains, and to produce a correspondingamide compound by bringing the transformant strain or the nitrilehydratase obtained therefrom into contact with the nitrile compound (seePatent Document 1).

On the other hand, when high activation of enzyme molecule itself can beachieved, the activity value of the enzyme preparation can be furtherenhanced.

Attempts have heretofore been made to search for a nitrile hydratasevariant with improved substrate specificity, enzyme stability or thelike by introducing mutation into a specific amino acid residue in theamino acid sequence of the nitrile hydratase without damaging itsactivity (see Patent Document 2 to 4).

Furthermore, a nitrile hydratase variant derived from Rhodococcusrhodochrous has been disclosed in Patent Documents 5 and 6.

RELATED DOCUMENT Patent Document

Patent Document 1: Japanese Patent Laid-open No. H9 (1997)-275978

Patent Document 2: Japanese Patent Laid-open No. 2004-194588

Patent Document 3: Japanese Patent Laid-open No. 2005-160403

Patent Document 4: WO 2004/056990

Patent Document 5: Japanese Patent Laid-open No. 2007-143409

Patent Document 6: Japanese Patent Laid-open No. 2008-253182

DISCLOSURE OF THE INVENTION

However, as compared to a wild nitrile hydratase disclosed in PatentDocument 1, specific mutants in which both of the initial reaction rateand enzyme stability are improved have not been known. It is expectedthat the production costs for producing the amide compound can bereduced by improving the initial reaction rate and enzyme stability atthe same time.

An object of the present invention is to provide a nitrile hydratasehaving high initial reaction rate and enzyme stability.

That is, the present invention is specified by matters described inbelow.

[1] A nitrile hydratase variant comprising substitution of at least oneamino acid with another amino acid to improve two or more properties ofnitrile hydratase by substitution of one or more and three or less aminoacids.

[2] The nitrile hydratase variant according to [1], wherein theproperties to be improved are the initial reaction rate and thermalstability.

[3] The nitrile hydratase variant according to [1] or [2], comprising anα-subunit defined in SEQ ID No: 1 in the Sequence Listing and aβ-subunit defined in SEQ ID No: 2 in the Sequence Listing, andsubstitution of at least one amino acid with another amino acid selectedfrom substitution sites of the amino acid consisting of the following(a) to (l):

(a) 92nd of α-subunit;

(b) 94th of α-subunit;

(c) 197th of α-subunit;

(d) 4th of β-subunit;

(e) 24th of β-subunit;

(f) 79th of β-subunit;

(g) 96th of β-subunit;

(h) 107th of β-subunit;

(i) 226th of β-subunit;

(j) 110th of β-subunit and 231st of β-subunit;

(k) 206th of β-subunit and 230th of β-subunit; and

(l) 13th of α-subunit, 27th of α-subunit and 110th of β-subunit.

[4] The nitrile hydratase variant according to [3], comprisingsubstitution of at least one amino acid with another amino acid selectedfrom substitution sites of the amino acid consisting of the following(m) to (u):

(m) in case of (b) or (g), 13th of α-subunit;

(n) in case of (b) or (h), 27th of α-subunit;

(o) (d) and (f);

(p) in case of (f), 230th of β-subunit;

(q) (a) and (i);

(r) in case of (i), 13th of α-subunit and 206th of β-subunit;

(s) in case of (a) and (d), 206th of β-subunit;

(t) in case of (c) and (h), 230th of β-subunit; and

(u) in case of (f), 230th of β-subunit and 231st of β-subunit.

[5] The nitrile hydratase variant according to [3], further comprisingsubstitution of at least one amino acid with another amino acid selectedfrom the group consisting of (a), (c), (f), (i), (h), 230th of theβ-subunit and 231st of the β-subunit in case of (e) is substituted withanother amino acid.

[6] The nitrile hydratase variant according to any one of [1] to [5],wherein Ile is substituted by Leu when 13th amino acid of the α-subunitis substituted,

Met is substituted by Ile when the 27th amino acid of the α-subunit issubstituted,

Asp is substituted by Glu when the 92nd amino acid of the α-subunit issubstituted,

Met is substituted by Ile when the 94th amino acid of the α-subunit issubstituted,

Gly is substituted by Cys when the 197th amino acid of the α-subunit issubstituted,

Val is substituted by Met when the 4th amino acid of the β-subunit issubstituted,

Val is substituted by Ile when the 24th amino acid of the β-subunit issubstituted,

His is substituted by Asn when the 79th amino acid of the β-subunit issubstituted,

Gln is substituted by Arg when the 96th amino acid of the β-subunit issubstituted,

Pro is substituted by Met when the 107th amino acid of the β-subunit issubstituted,

Glu is substituted by Asn when the 110th amino acid of the β-subunit issubstituted,

Pro is substituted by Leu when the 206th amino acid of the β-subunit issubstituted,

Val is substituted by Ile when the 226th amino acid of the β-subunit issubstituted,

Ala is substituted by Glu when the 230th amino acid of the β-subunit issubstituted, and

Ala is substituted by Val when the 231st amino acid of the β-subunit issubstituted.

[7] The nitrile hydratase variant according to any one of [3] to [6],further comprising substitution of at least one amino acid selected fromsubstitutions of the amino acid consisting of the following (aa) to(br):

(aa) 36th Thr in the α-subunit is substituted by Met and 126th Phe inthe α-subunit is substituted by Tyr;

(ab) 148th Gly in the α-subunit is substituted by Asp and 204th Val inthe α-subunit is substituted by Arg;

(ac) 51st Phe in the β-subunit is substituted by Val and 108th Glu inthe β-subunit is substituted by Asp;

(ad) 118th Phe in the β-subunit is substituted by Val and 200th Ala inthe β-subunit is substituted by Glu;

(ae) 160th Arg in the β-subunit is substituted by Trp and 186th Leu inthe β-subunit is substituted by Arg;

(af) 6th Leu in the α-subunit is substituted by Thr, 36th Thr in theα-subunit is substituted by Met, and 126th Phe in the α-subunit issubstituted by Tyr;

(ag) 19th Ala in the α-subunit is substituted by Val, 71st Arg in theα-subunit is substituted by His, and 126th Phe in the α-subunit issubstituted by Tyr;

(ah) 36th Thr in the α-subunit is substituted by Met, 148th Gly in theα-subunit is substituted by Asp, and 204th Val in the α-subunit issubstituted by Arg;

(ai) 10th Thr in the β-subunit is substituted by Asp, 118th Phe in theβ-subunit is substituted by Val, and 200th Ala in the β-subunit issubstituted by Glu;

(aj) 37th Phe in the β-subunit is substituted by Leu, 108th Glu in theβ-subunit is substituted by Asp, and 200th Ala in the β-subunit issubstituted by Glu;

(ak) 37th Phe in the β-subunit is substituted by Val, 108th Glu in theβ-subunit is substituted by Asp, and 200th Ala in the β-subunit issubstituted by Glu;

(al) 41st Phe in the β-subunit is substituted by Ile, 51st Phe in theβ-subunit is substituted by Val, and 108th Glu in the β-subunit issubstituted by Asp;

(am) 46th Met in the β-subunit is substituted by Lys, 108th Glu in theβ-subunit is substituted by Arg, and 212th Ser in the β-subunit issubstituted by Tyr;

(an) 48th Leu in the β-subunit is substituted by Val, 108th Glu in theβ-subunit is substituted by Arg, and 212th Ser in the β-subunit issubstituted by Tyr;

(ao) 127th Leu in the β-subunit is substituted by Ser, 160th Arg in theβ-subunit is substituted by Trp, and 186th Leu in the β-subunit issubstituted by Arg;

(ap) 6th Leu in the α-subunit is substituted by Thr, 19th Ala in theα-subunit is substituted by Val, 126th Phe in the α-subunit issubstituted by Tyr, 46th Met in the β-subunit is substituted by Lys,108th Glu in the β-subunit is substituted by Arg, and 212th Ser in theβ-subunit is substituted by Tyr;

(aq) 6th Leu in the α-subunit is substituted by Thr, 19th Ala in theα-subunit is substituted by Val, 126th Phe in the α-subunit issubstituted by Tyr, 48th Leu in the β-subunit is substituted by Val,108th Glu in the β-subunit is substituted by Arg, and 212th Ser in theβ-subunit is substituted by Tyr;

(ar) 6th Leu in the α-subunit is substituted by Ala, 19th Ala in theα-subunit is substituted by Val, 126th Phe in the α-subunit issubstituted by Tyr, 127th Leu in the β-subunit is substituted by Ser,160th Arg in the β-subunit is substituted by Trp, and 186th Leu in theβ-subunit is substituted by Arg;

(as) 6th Leu in the α-subunit is substituted by Thr, 36th Thr in theα-subunit is substituted by Met, 126th Phe in the α-subunit issubstituted by Tyr, 10th Thr in the β-subunit is substituted by Asp,118th Phe in the β-subunit is substituted by Val, and 200th Ala in theβ-subunit is substituted by Glu;

(at) 19th Ala in the α-subunit is substituted by Val, 71st Arg in theα-subunit is substituted by His, 126th Phe in the α-subunit issubstituted by Tyr, 37th Phe in the β-subunit is substituted by Leu,108th Glu in the β-subunit is substituted by Asp, and 200th Ala in theβ-subunit is substituted by Glu;

(au) 19th Ala in the α-subunit is substituted by Val, 71st Arg in theα-subunit is substituted by His, 126th Phe in the α-subunit issubstituted by Tyr, 37th Phe in the β-subunit is substituted by Val,108th Glu in the β-subunit is substituted by Asp, and 200th Ala in theβ-subunit is substituted by Glu;

(av) 36th Thr in the α-subunit is substituted by Met, 148th Gly in theα-subunit is substituted by Asp, 204th Val in the α-subunit issubstituted by Arg, 41st Phe in the β-subunit is substituted by Ile,51st Phe in the β-subunit is substituted by Val, and 108th Glu in theβ-subunit is substituted by Asp;

(aw) 148th Gly in the α-subunit is substituted by Asp, 204th Val in theα-subunit is substituted by Arg, 108th Glu in the β-subunit issubstituted by Asp, and 200th Ala in the β-subunit is substituted byGlu;

(ax) 36th Thr in the α-subunit is substituted by Gly and 188th Thr inthe α-subunit is substituted by Gly;

(ay) 36th Thr in the α-subunit is substituted by Ala and 48th Asn in theα-subunit is substituted by Gln;

(az) 48th Asn in the α-subunit is substituted by Glu and 146th Arg inthe β-subunit is substituted by Gly;

(ba) 36th Thr in the α-subunit is substituted by Trp and 176th Tyr inthe β-subunit is substituted by Cys;

(bb) 176th Tyr in the β-subunit is substituted by Met and 217th Asp inthe β-subunit is substituted by Gly;

(bc) 36th Thr in the α-subunit is substituted by Ser, and 33rd Ala inthe β-subunit is substituted by Val;

(bd) 176th Tyr in the β-subunit is substituted by Ala and 217th Asp inthe β-subunit is substituted by Val;

(be) 40th Thr in the β-subunit is substituted by Val and 218th Cys inthe β-subunit is substituted by Met;

(bf) 33rd Ala in the β-subunit is substituted by Met and 176th Tyr inthe β-subunit is substituted by Thr;

(bg) 40th Thr in the β-subunit is substituted by Leu and 217th Asp inthe β-subunit is substituted by Leu;

(bh) 40th Thr in the β-subunit is substituted by Ile and 61st Ala in theβ-subunit is substituted by Val;

(bi) 61st Ala in the β-subunit is substituted by Thr and 218th Cys inthe β-subunit is substituted by Ser;

(bj) 112th Lys in the β-subunit is substituted by Val and 217th Asp inthe β-subunit is substituted by Met;

(bk) 61st Ala in the β-subunit is substituted by Trp and 217th Asp inthe β-subunit is substituted by His;

(bl) 61st Ala in the β-subunit is substituted by Leu and 112th Lys inthe β-subunit is substituted by Ile;

(bm) 146th Arg in the β-subunit is substituted by Gly and 217th Asp inthe β-subunit is substituted by Ser;

(bn) 171st Lys in the β-subunit is substituted by Ala and 217th Asp inthe β-subunit is substituted by Thr;

(bo) 150th Ala in the β-subunit is substituted by Ser and 217th Asp inthe β-subunit is substituted by Cys;

(bp) 61st Ala in the β-subunit is substituted by Gly and 150th Ala inthe β-subunit is substituted by Asn;

(bq) 61st Ala in the β-subunit is substituted by Ser and 160th Arg inthe β-subunit is substituted by Met; and

(br) 160th Arg in the β-subunit is substituted by Cys and 168th Thr inthe β-subunit is substituted by Glu.

[8] A gene encoding the nitrile hydratase variant according to any oneof [1] to [7].

[9] A gene encoding a nitrile hydratase variant having a gene encodingthe α-subunit defined in SEQ ID No: 3 in the Sequence Listing and a geneencoding the β-subunit defined in SEQ ID No: 4 in the Sequence Listing,comprising substitution of at least one base selected from substitutionsites of the base consisting of the following (a) to (l):

(a) 274th to 276th of the base sequence of SEQ ID No: 3;

(b) 280th to 282nd of the base sequence of SEQ ID No: 3;

(c) 589th to 591st of the base sequence of SEQ ID No: 3;

(d) 10th to 12th of the base sequence of SEQ ID No: 4;

(e) 69th to 71st of the base sequence of SEQ ID No: 4;

(f) 235th to 237th of the base sequence of SEQ ID No: 4;

(g) 286th to 288th of the base sequence of SEQ ID No: 4;

(h) 319th to 321st of the base sequence of SEQ ID No: 4;

(i) 676th to 678th of the base sequence of SEQ ID No: 4;

(j) 328th to 330th of the base sequence of SEQ ID No: 4 and 691st to693rd of the base sequence of SEQ ID No: 4;

(k) 616th to 618th of the base sequence of SEQ ID No: 4, and 688th to690th of the base sequence of SEQ ID No: 4; and

(l) 37th to 39th of the base sequence of SEQ ID No: 3, 79th to 81st ofthe base sequence of SEQ ID No: 3, and 328th to 330th of the basesequence of SEQ ID No: 4.

[10] The gene encoding a nitrile hydratase variant according to [9],further comprising substitution of at least one base selected fromsubstitution sites of the base consisting of the following (m) to (u):

(m) in case of (b) or (g), 37th to 39th of the base sequence of SEQ IDNo: 3;

(n) in case of (b) or (h), 79th to 81st of the base sequence of SEQ IDNo: 3;

(o) (d) and (f);

(p) in case of (f), 688th to 690th of the base sequence of SEQ ID No: 4;

(q) (a) and (i);

(r) in case of (i), 37th to 39th of the base sequence of SEQ ID No: 3and 616th to 618th of the base sequence of SEQ ID No: 4;

(s) in case of (a) and (d), 616th to 618th of the base sequence of SEQID No: 4;

(t) in case of (c) and (h), 688th to 690th of the base sequence of SEQID No: 4; and

(u) in case of (f), 688th to 690th of the base sequence of SEQ ID No: 4and 691st to 693rd of the base sequence of SEQ ID No: 4.

[11] The gene encoding a nitrile hydratase variant according to [9],further comprising substitution of at least one base with another baseselected from substitution sites of the base consisting of (a), (c),(f), (i), (h), 688th to 690th of the base sequence of SEQ ID No: 4, and691st to 693rd of the base sequence of SEQ ID No: 4, in case of (e), aresubstituted with another base.

[12] The gene encoding a nitrile hydratase variant according to any oneof [9] to [11], wherein ATC is substituted by CTC when 37th to 39th ofthe base sequence of SEQ ID No: 3 are substituted by another base,

ATG is substituted by ATC when 79th to 81th of the base sequence of SEQID No: 3 are substituted by another base,

GAC is substituted by GAG when 274th to 276th of the base sequence ofSEQ ID No: 3 are substituted by another base,

ATG is substituted by ATC when 280th to 282th of the base sequence ofSEQ ID No: 3 are substituted by another base,

GGC is substituted by TGC when 589th to 591th of the base sequence ofSEQ ID No: 3 are substituted by another base,

GTG is substituted by ATG when 10th to 12th of the base sequence of SEQID No: 4 are substituted by another base,

GTC is substituted by ATC when 69th to 71th of the base sequence of SEQID No: 4 are substituted by another base,

CAC is substituted by AAC when 235th to 237th of the base sequence ofSEQ ID No: 4 are substituted by another base,

CAG is substituted by CGT when 286th to 288th of the base sequence ofSEQ ID No: 4 are substituted by another base,

CCC is substituted by ATG when 319th to 321st of the base sequence ofSEQ ID No: 4 are substituted by another base,

GAG is substituted by AAC when 328th to 330th of the base sequence ofSEQ ID No: 4 are substituted by another base,

CCG is substituted by CTG when 616th to 618th of the base sequence ofSEQ ID No: 4 are substituted by another base,

GTC is substituted by ATC when 676th to 678th of the base sequence ofSEQ ID No: 4 are substituted by another base,

GCG is substituted by GAG when 688th to 690th of the base sequence ofSEQ ID No: 4 are substituted by another base, and

GCC is substituted by GTC when 691th to 693th of the base sequence ofSEQ ID No: 4 are substituted by another base.

[13] The gene encoding a nitrile hydratase variant according to any oneof [9] to [12], comprising substitution of at least one base selectedfrom substitution sites of the base consisting of the following (aa) to(br), and having the nitrile hydratase activity:

(aa) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by ATG, and 376th to 378th TTC of the base sequence of SEQID No: 3 are substituted by TAC;

(ab) 442nd to 444th GGC of the base sequence of SEQ ID No: 3 aresubstituted by GAC, and 610th to 612th GTC of the base sequence of SEQID No: 3 are substituted by CGC;

(ac) 151st to 153rd TTC of the base sequence of SEQ ID No: 4 aresubstituted by GTC, and 322nd to 324th GAG of the base sequence of SEQID No: 4 are substituted by GAT;

(ad) 352nd to 354th TTC of the base sequence of SEQ ID No: 4 aresubstituted by GTC, and 598th to 600th GCC of the base sequence of SEQID No: 4 are substituted by GAG;

(ae) 478th to 480th CGG of the base sequence of SEQ ID No: 4 aresubstituted by TGG, and 556th to 558th CTG of the base sequence of SEQID No: 4 are substituted by CGG;

(af) 16th to 18th CTG of the base sequence of SEQ ID No: 3 aresubstituted by ACG, 106th to 108th ACG of the base sequence of SEQ IDNo: 3 are substituted by ATG, and 376th to 378th TTC of the basesequence of SEQ ID No: 3 are substituted by TAC;

(ag) 55th to 57th GCG of the base sequence of SEQ ID No: 3 aresubstituted by GTG, 211th to 213th CGT of the base sequence of SEQ IDNo: 3 are substituted by CAT, and 376th to 378th TTC of the basesequence of SEQ ID No: 3 are substituted by TAC;

(ah) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by ATG, 442nd to 444th GGC of the base sequence of SEQ IDNo: 3 are substituted by GAC, and 610th to 612th GTC of the basesequence of SEQ ID No: 3 are substituted by CGC;

(ai) 28th to 30th ACC of the base sequence of SEQ ID No: 4 aresubstituted by GAC, 352nd to 354th TTC of the base sequence of SEQ IDNo: 4 are substituted by GTC, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG;

(aj) 109th to 111th TTC of the base sequence of SEQ ID No: 4 aresubstituted by CTC, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by GAT, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG;

(ak) 109th to 111th TTC of the base sequence of SEQ ID No: 4 aresubstituted by GTC, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by GAT, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG;

(al) 121st to 123rd TTC of the base sequence of SEQ ID No: 4 aresubstituted by ATC, 151st to 153rd TTC of the base sequence of SEQ IDNo: 4 are substituted by GTC, and 322nd to 324th GAG of the basesequence of SEQ ID No: 4 are substituted by GAT;

(am) 136th to 138th ATG of the base sequence of SEQ ID No: 4 aresubstituted by AAG, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by CGG, and 634th to 636th TCC of the basesequence of SEQ ID No: 4 are substituted by TAC;

(an) 142nd to 144th CTG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by CGG, and 634th to 636th TCC of the basesequence of SEQ ID No: 4 are substituted by TAC;

(ao) 379th to 381st CTG of the base sequence of SEQ ID No: 4 aresubstituted by TCG, 478th to 480th CGG of the base sequence of SEQ IDNo: 4 are substituted by TGG, and 556th to 558th CTG of the basesequence of SEQ ID No: 4 are substituted by CGG;

(ap) 16th to 18th CTG of the base sequence of SEQ ID No: 3 aresubstituted by ACG, 55th to 57th GCG of the base sequence of SEQ ID No:3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQID No: 3 are substituted by TAC, 136th to 138th ATG of the base sequenceof SEQ ID No: 4 are substituted by AAG, 322nd to 324th GAG of the basesequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCCof the base sequence of SEQ ID No: 4 are substituted by TAC;

(aq) 16th to 18th CTG of the base sequence of SEQ ID No: 3 aresubstituted by ACG, 55th to 57th GCG of the base sequence of SEQ ID No:3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQID No: 3 are substituted by TAC, 142nd to 144th CTG of the base sequenceof SEQ ID No: 4 are substituted by GTG, 322nd to 324th GAG of the basesequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCCof the base sequence of SEQ ID No: 4 are substituted by TAC;

(ar) 16th to 18th CTG of the base sequence of SEQ ID No: 3 aresubstituted by GCG, 55th to 57th GCG of the base sequence of SEQ ID No:3 are substituted by GTG, 376th to 378th TTC of the base sequence of SEQID No: 3 are substituted by TAC, 379th to 381st CTG of the base sequenceof SEQ ID No: 4 are substituted by TCG, 478th to 480th CGG of the basesequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTGof the base sequence of SEQ ID No: 4 are substituted by CGG;

(as) 16th to 18th CTG of the base sequence of SEQ ID No: 3 aresubstituted by ACG, 106th to 108th ACG of the base sequence of SEQ IDNo: 3 are substituted by ATG, 376th to 378th TTC of the base sequence ofSEQ ID No: 3 are substituted by TAC, 28th to 30th ACC of the basesequence of SEQ ID No: 4 are substituted by GAC, 352nd to 354th TTC ofthe base sequence of SEQ ID No: 4 are substituted by GTC, and 598th to600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;

(at) 55th to 57th GCG of the base sequence of SEQ ID No: 3 aresubstituted by GTG, 211th to 213th CGT of the base sequence of SEQ IDNo: 3 are substituted by CAT, 376th to 378th TTC of the base sequence ofSEQ ID No: 3 are substituted by TAC, 109th to 111th TTC of the basesequence of SEQ ID No: 4 are substituted by CTC, 322nd to 324th GAG ofthe base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;

(au) 55th to 57th GCG of the base sequence of SEQ ID No: 3 aresubstituted by GTG, 211th to 213th CGT of the base sequence of SEQ IDNo: 3 are substituted by CAT, 376th to 378th TTC of the base sequence ofSEQ ID No: 3 are substituted by TAC, 109th to 111th TTC of the basesequence of SEQ ID No: 4 are substituted by GTC, 322nd to 324th GAG ofthe base sequence of SEQ ID No: 4 are substituted by GAT, and 598th to600th GCC of the base sequence of SEQ ID No: 4 are substituted by GAG;

(av) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by ATG, 442nd to 444th GGC of the base sequence of SEQ IDNo: 3 are substituted by GAC, 610th to 612th GTC of the base sequence ofSEQ ID No: 3 are substituted by CGC, 121st to 123rd TTC of the basesequence of SEQ ID No: 4 are substituted by ATC, 151st to 153rd TTC ofthe base sequence of SEQ ID No: 4 are substituted by GTC, and 322nd to324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT;

(aw) 442nd to 444th GGC of the base sequence of SEQ ID No: 3 aresubstituted by GAC, 610th to 612th GTC of the base sequence of SEQ IDNo: 3 are substituted by CGC, 322nd to 324th GAG of the base sequence ofSEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG;

(ax) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by GGG, and 562nd to 564th ACC of the base sequence of SEQID No: 3 are substituted by GGC;

(ay) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by GCG, and 142nd to 144th AAC of the base sequence of SEQID No: 3 are substituted by CAA;

(az) 142nd to 144th AAC of the base sequence of SEQ ID No: 3 aresubstituted by GAA, and 436th to 438th CGG of the base sequence of SEQID No: 4 are substituted by GGG;

(ba) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by TGG, and 526th to 528th TAC of the base sequence of SEQID No: 4 are substituted by TGC;

(bb) 526th to 528th TAC of the base sequence of SEQ ID No: 4 aresubstituted by ATG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by GGC;

(bc) 106th to 108th ACG of the base sequence of SEQ ID No: 3 aresubstituted by TCG, and 97th to 99th GCG of the base sequence of SEQ IDNo: 4 are substituted by GTG;

(bd) 526th to 528th TAC of the base sequence of SEQ ID No: 4 aresubstituted by GCC, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by GTC;

(be) 118th to 120th ACG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, and 652nd to 654th TGC of the base sequence of SEQID No: 4 are substituted by ATG;

(bf) 97th to 99th GCG of the base sequence of SEQ ID No: 4 aresubstituted by ATG, and 526th to 528th TAC of the base sequence of SEQID No: 4 are substituted by ACC;

(bg) 118th to 120th ACG of the base sequence of SEQ ID No: 4 aresubstituted by CTG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by CTC;

(bh) 118th to 120th ACG of the base sequence of SEQ ID No: 4 aresubstituted by ATT, and 181st to 183rd GCC of the base sequence of SEQID No: 4 are substituted by GTC;

(bi) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by ACG, and 652nd to 654th TGC of the base sequence of SEQID No: 4 are substituted by TCC;

(bj) 334th to 336th AAG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by ATG;

(bk) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by TGG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by CAC;

(bl) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by CTC, and 334th to 336th AAG of the base sequence of SEQID No: 4 are substituted by ATT;

(bm) 436th to 438th CGG of the base sequence of SEQ ID No: 4 aresubstituted by GGG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by AGC;

(bn) 511th to 513th AAG of the base sequence of SEQ ID No: 4 aresubstituted by GCG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by ACC;

(bo) 448th to 450th GCG of the base sequence of SEQ ID No: 4 aresubstituted by TCG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by TGT;

(bp) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by GGC, and 448th to 450th GCG of the base sequence of SEQID No: 4 are substituted by AAT;

(bq) 181st to 183rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by TCG, and 478th to 480th CGG of the base sequence of SEQID No: 4 are substituted by ATG; and

(br) 478th to 480th CGG of the base sequence of SEQ ID No: 4 aresubstituted by TGT, and 502nd to 504th ACG of the base sequence of SEQID No: 4 are substituted by GAG.

[14] A linked DNA comprising further DNA containing a promoter sequencenecessary for the expression of the gene in the upstream region of the5′-terminal of the gene encoding a nitrile hydratase variant accordingto any one of [9] to [13], and a ribosome binding sequence contained inSEQ ID No: 7 in the downstream region of the 3′-terminal of thepromoter.

[15] A plasmid comprising the DNA according to [14].

[16] A transformant obtained by transformation of a host cell using theplasmid according to [15].

[17] A method for producing a nitrile hydratase variant, comprisingcultivating the transformant according to [16] in a culture medium andproducing a nitrile hydratase variant based on the nitrile hydratasegene carried by the plasmid in the transformant.

According to the present invention, a nitrile hydratase composed of anα-subunit defined in SEQ ID No: 1 in the Sequence Listing and aβ-subunit defined in SEQ ID No: 2 in the Sequence Listing comprisessubstitution of at least one amino acid with another amino acid,selected from substitution sites of the amino acid consisting of theabove (a) to (l). Thus, both of the initial reaction rate and enzymestability of the nitrile hydratase are improved, so that the activityvalue in a unit weight of the enzyme preparation can be increased, andat the same time the risk of enzyme deactivation due to temperaturevariation or the like for the industrial use can be reduced.Accordingly, the amide compound can be stably produced with a smalleramount of the enzyme, so that the production costs for producing theamide compound can be reduced.

According to the present invention, it is possible to provide a novelnitrile hydratase variant in which the initial reaction rate and enzymestability are improved than those of the wild nitrile hydratase, and toreduce the production costs for the enzyme in the total production costsfor producing the amide compound.

DESCRIPTION OF EMBODIMENTS

The above and other objects, features and advantages will be moreapparent from the following description of the preferred embodiments.The present invention will be described in more detail below.

The nitrile hydratase variant of the present invention comprisessubstitution of at least one amino acid with another amino acid toimprove two or more properties of the nitrile hydratase by thesubstitution of one or more three or less amino acids.

The term “properties” to be improved in the nitrile hydratase variant ofthe present invention refer to properties relating to the reactionitself for hydrating a nitrile group to convert it into an amide group,and enzyme stability. The term “properties” relating to the reactionitself refer to the activity of the enzyme, the substrate specificity,Vmax, Km, and the initial reaction rate. The enzyme stability includesthermal stability, stability against the substrate, and stabilityagainst the product.

The nitrile hydratase variant of the present invention preferablycomprises substitution of at least one amino acid with another aminoacid to improve properties of the thermophilic bacteria-derived nitrilehydratase. As the thermophilic bacteria, suitably used are thosebelonging to the genus Psuedonocardia. A specific example includesPsuedonocardia thermophila.

More specifically, the nitlile hydratase variant includes at least oneamino acid substituted with another amino acid, selected fromsubstitution sites of (a) to (l) as shown in Table I, in the nitrilehydratase consisting of the α-subunit defined in SEQ ID No: 1 in theSequence Listing and the β-subunit defined in SEQ ID No: 2 in theSequence Listing. Thus, the nitrile hydratase variant of the presentinvention is provided with higher initial reaction rate and enzymestability than the wild nitrile hydratase as described in PatentDocument 1.

TABLE I Before After SEQ ID No. No. Substitution Substitution (a) 1 92Asp Glu (b) 1 94 Met Ile (c) 1 197 Gly Cys (d) 2 4 Val Met (e) 2 24 ValIle (f) 2 79 His Asn (g) 2 96 Gln Arg (h) 2 107 Pro Met (i) 2 226 ValIle (j) 2 110 Glu Asn 2 231 Ala Val (k) 2 206 Pro Leu 2 230 Ala Glu (l)1 13 Ile Leu 1 27 Met Ile 2 110 Glu Asn

A plurality of substitutions of the amino acid of (a) to (l) shown inTable I may be combined, or may be combined with substitutions of theamino acid at the different sites other than (a) to (l). For example, incase of (e), at least one amino acid selected from the group consistingof (a), (c), (f), (i), (h), 230th of the β-subunit and 231st of theβ-subunit may be substituted with another amino acid. Examples ofsubstitution of the amino acid which can be combined with (a) to (l)include those in Table II.

TABLE II Before After SEQ ID No. No. Substitution Substitution (m-1) 113 Ile Leu 1 94 Met Ile (m-2) 1 13 Ile Leu 2 96 Gln Arg (n-1) 1 27 MetIle 1 94 Met Ile (n-2) 1 27 Met Ile 2 107 Pro Met (o) 2 4 Val Met 2 79His Asn (p) 2 79 His Asn 2 230 Ala Glu (q) 1 92 Asp Glu 2 226 Val Ile(r) 1 13 Ile Leu 2 206 Pro Leu 2 226 Val Ile (s) 1 92 Asp Glu 2 4 ValMet 2 206 Pro Leu (t) 1 197 Gly Cys 2 107 Pro Met 2 230 Ala Glu (u) 2 79His Asn 2 230 Ala Glu 2 231 Ala Val (v) 1 92 Asp Glu 2 24 Val Ile 2 226Val Ile (w) 1 197 Gly Cys 2 24 Val Ile 2 107 Pro Met 2 230 Ala Glu (x) 224 Val Ile 2 79 His Asn 2 230 Ala Glu 2 231 Ala Val

The nitrile hydratase variant of the present invention may furthercomprise mutation in any one nitrile hydratase variant of the above (a)to (x) at sites (aa) to (br) of the amino acid of the nitrile hydrataseof SEQ ID Nos: 1 and 2 as shown in Table III.

TABLE III Before After SEQ ID No. No. Substitution Substitution (aa) 136 Thr Met 1 126 Phe Tyr (ab) 1 148 Gly Asp 1 204 Val Arg (ac) 2 51 PheVal 2 108 Glu Asp (ad) 2 118 Phe Val 2 200 Ala Glu (ae) 2 160 Arg Trp 2186 Leu Arg (af) 1 6 Leu Thr 1 36 Thr Met 1 126 Phe Tyr (ag) 1 19 AlaVal 1 71 Arg His 1 126 Phe Tyr (ah) 1 36 Thr Met 1 148 Gly Asp 1 204 ValArg (ai) 2 10 Thr Asp 2 118 Phe Val 2 200 Ala Glu (aj) 2 37 Phe Leu 2108 Glu Asp 2 200 Ala Glu (ak) 2 37 Phe Val 2 108 Glu Asp 2 200 Ala Glu(al) 2 41 Phe Ile 2 51 Phe Val 2 108 Glu Asp

TABLE III-1 (am) 2 46 Met Lys 2 108 Glu Arg 2 212 Ser Tyr (an) 2 48 LeuVal 2 108 Glu Arg 2 212 Ser Tyr (ao) 2 127 Leu Ser 2 160 Arg Trp 2 186Leu Arg (ap) 1 6 Leu Thr 1 19 Ala Val 1 126 Phe Tyr 2 46 Met Lys 2 108Glu Arg 2 212 Ser Tyr (aq) 1 6 Leu Thr 1 19 Ala Val 1 126 Phe Tyr 2 48Leu Val 2 108 Glu Arg 2 212 Ser Tyr (ar) 1 6 Leu Ala 1 19 Ala Val 1 126Phe Tyr 2 127 Leu Ser 2 160 Arg Trp 2 186 Leu Arg (as) 1 6 Leu Thr 1 36Thr Met 1 126 Phe Tyr 2 10 Thr Asp 2 118 Phe Val 2 200 Ala Glu

TABLE III-2 (at) 1 19 Ala Val 1 71 Arg His 1 126 Phe Tyr 2 37 Phe Leu 2108 Glu Asp 2 200 Ala Glu (au) 1 19 Ala Val 1 71 Arg His 1 126 Phe Tyr 237 Phe Val 2 108 Glu Asp 2 200 Ala Glu (av) 1 36 Thr Met 1 148 Gly Asp 1204 Val Arg 2 41 Phe Ile 2 51 Phe Val 2 108 Glu Asp (aw) 1 148 Gly Asp 1204 Val Arg 2 108 Glu Asp 2 200 Ala Glu (ax) 1 36 Thr Gly 1 188 Thr Gly(ay) 1 36 Thr Ala 1 48 Asn Gln (az) 1 48 Asn Glu 2 146 Arg Gly (ba) 1 36Thr Trp 2 176 Tyr Cys (bb) 2 176 Tyr Met 2 217 Asp Gly (bc) 1 36 Thr Ser2 33 Ala Val

TABLE III-3 (bd) 2 176 Tyr Ala 2 217 Asp Val (be) 2 40 Thr Val 2 218 CysMet (bf) 2 33 Ala Met 2 176 Tyr Thr (bg) 2 40 Thr Leu 2 217 Asp Leu (bh)2 40 Thr Ile 2 61 Ala Val (bi) 2 61 Ala Thr 2 218 Cys Ser (bj) 2 112 LysVal 2 217 Asp Met (bk) 2 61 Ala Trp 2 217 Asp His (bl) 2 61 Ala Leu 2112 Lys Ile (bm) 2 146 Arg Gly 2 217 Asp Ser (bn) 2 171 Lys Ala 2 217Asp Thr (bo) 2 150 Ala Ser 2 217 Asp Cys (bp) 2 61 Ala Gly 2 150 Ala Asn(bq) 2 61 Ala Ser 2 160 Arg Met (br) 2 160 Arg Cys 2 168 Thr Glu

In the present invention, the term “nitrile hydratase activity” refersto the nitrile-hydrating activity to convert a nitrile group of variouscompounds to an amide group by hydration, and more preferably refers tothe activity to convert acrylonitrile to acrylamide.

In the present invention, the term “improved nitrile hydratase activity”refers to improvement of the initial reaction rate. The “initialreaction rate” in the present invention may be confirmed in thefollowing manner. First, a nitrile hydratase preparation is added to a50 mM Tris-HCl aqueous solution (pH 8.0) containing 2.5% (v/v) ofacrylonitrile as a substrate. In place of the nitrile hydratasepreparation, a microorganism cell, a culture or a crude purificationproduct of the nitrile hydratase may be used. After the addition of thenitrile hydratase, the reaction is carried out at 20 degrees centigradefor 15 minutes. 1M phosphoric acid is added to the reaction solution tostop the reaction, and the produced acrylamide is quantitativelyanalyzed. The amount of acrylamide may be measured through HPLCanalysis.

The term “improvement of the initial reaction rate” in the presentinvention refers to significant improvement of the initial reaction rateas compared to the wild nitrile hydratase and conventionally knownnitrile hydratase variant, and specifically refers to improvement of notless than 1.2 times.

The term “improvement of enzyme stability” in the present inventionrefers to improvement of thermal stability of the nitrile hydratase. Thenitrile hydratase with improved thermal stability is expected toincrease stability against stress other than heating, i.e., stabilityagainst an organic solvent, a high-concentration substrate or a productas well, because structural stability of a protein is considered to bestrengthened.

The term “thermal stability of the enzyme” in the present invention maybe confirmed in the following manner. First, a nitrile hydratasepreparation is heated at 60 degrees centigrade for 2 hours, and then thetemperature is returned to 20 degrees centigrade, and a 50 mM Tris-HClaqueous solution (pH 8.0) containing 2.5% (v/v) of acrylonitrile as asubstrate is added thereto. In place of the nitrile hydratasepreparation, a microorganism cell, a culture or a crude purificationproduct of the nitrile hydratase may be used. The heated nitrilehydratase and substrate are mixed together, and then reacted at 20degrees centigrade for 15 minutes to measure the initial reaction rate.

The term “improvement of thermal stability of the enzyme” in the presentinvention refers to significant improvement of the initial reaction rateafter heating as compared to the wild nitrile hydratase andconventionally known nitrile hydratase variant heated in the samemanner, and specifically refers to improvement of not less than 1.2times.

As the wild nitrile hydratase in the present invention, preferably usedis Pseudonocardia thermophila-derived nitrile hydratase as disclosed inPatent Document 1. As the plasmid expressing a large number of the wildnitrile hydratase in the transformant and a transformant straintransformed with the plasmid, there may be cited MT-10822 (depositedwith the International Patent Organism Depositary at the NationalInstitute of Advanced Industrial Science and Technology, 1-1-1 Higashi,Tsukuba-shi, Ibaraki-ken, Japan, under the deposit number FERM BP-5785,as of Feb. 7, 1996). As the conventionally known nitrile hydratasevariant in the present invention, there may be cited the nitrilehydratase variant described in Patent Documents 1 to 4.

The nitrile hydratase variant of the present invention has the followingproperties in addition to improvement of the nitrile hydratase activity.The enzyme comprises a dimer having the α-subunit and the β-subunitwhich are in association as the fundamental structural unit, and thedimers are further associated to form tetramers. 111th cysteine residueof the α-subunit undergoes a post-translational modification in acysteine sulfinic acid (Cys-SOOH), while 113th cysteine residueundergoes a post-translational modification in a cysteine sulfenic acid(Cys-SOH). A polypeptide chain of the α-subunit is bonded to a cobaltatom via the modified amino acid residue to form an active center. Thereaction may be preferably carried out in the temperature range of 0 to60 degrees centigrade, while pH during the reaction is usually selectedin the range of 4 to 10 and preferably in the range of 6 to 9.

The nitrile hydratase variant of the present invention may be producedin the following manner.

First, a plasmid containing DNA encoding the nitrile hydratase variantis prepared, and a transformant or a transformant strain is obtained bytransforming an arbitrary host cell using the plasmid. Subsequently, thenitrile hydratase variant is produced by cultivating the above-mentionedtransformant or transformant strain.

The gene encoding a wild nitrile hydratase comprises a base sequencedefined in SEQ ID No: 3 in the Sequence Listing and a base sequencedefined in SEQ ID No: 4 in the Sequence Listing. The base sequencedefined in SEQ ID No: 3 in the Sequence Listing corresponds to the aminoacid sequence consisting of SEQ ID No: 1 in the Sequence Listing, whilethe base sequence defined in SEQ ID No: 4 in the Sequence Listingcorresponds to the amino acid sequence consisting of SEQ ID No: 2 in theSequence Listing. DNA encoding the nitrile hydratase variant can beobtained by performing base substitution of the base sequence defined inSEQ ID No: 3 and/or SEQ ID No: 4. Specifically, substitutions of theamino acid of (a) to (l) shown in Table I may be realized by basesubstitution, as shown in Table IV-1.

TABLE IV-1 Sequence ID Before No. No. Substitution After Substitution(a) 3 274~276 GAC GAA, GAG (b) 3 280~282 ATG ATT, ATC, ATA (c) 3 589~591GGC TGT, TGC (d) 4 10~12 GTG ATG (e) 4 69~71 GTC ATT, ATC, ATA (f) 4235~237 CAC AAT, AAC (g) 4 286~288 CAG CGT, CGG, CGA, CGG, AGA, AGG (h)4 319~321 CCC ATG (i) 4 676~678 GTC ATT, ATC, ATA (j) 4 328~330 GAG AAT,AAC 4 691~693 GCC GTT, GTG, GTA, GTG (k) 4 616~618 CCG TTA, TTG, CTT,CTC, CTA, CTG 4 688~690 GCG GAA, GAG (l) 3 37~39 ATC TTA, TTG, CTT, CTC,CTA, GTG 3 79~81 ATG ATT, ATC, ATA 4 328~330 GAG AAT, AAC

Furthermore, substitutions of the amino acid illustrated in Table II maybe realized by base substitution, as shown in Table IV-2.

TABLE IV-2 Sequence Before ID No. No. Substitution After Substitution(m-1) 3 37~39 ATC TTA TTG, CTT, CTC, CTA, CTG 3 280~282 ATG ATT, ATC,ATA (m-2) 3 37~39 ATC TTA, TTG, CTT, CTC, CTA, CTG 4 286~288 CAG CGT,CGC, CGA, CGG, AGA AGG (n-1) 3 79~81 ATG ATT, ATC, ATA 4 280~282 ATGATT, ATC, ATA (n-2) 3 79~81 ATG ATT, ATC, ATA 4 319~321 CCC ATG (o) 410~12 GTG ATG 4 235~237 CAC AAT, AAC (p) 4 235~237 CAC AAT, AAC 4688~690 GCG GAA, GAG (q) 3 274~276 GAC GAA, GAG 4 676~678 GTC ATT, ATC,ATA (r) 3 37~39 ATC TTA, TTG, CTT, CTC, CTA CTG 4 616~618 CCG TTA, TTG,CTT, CTC, CTA, CTG 4 676~678 GTC ATT, ATC, ATA (s) 3 274~276 GAC GAA,GAG 4 10~12 GTG ATG 4 616~618 CCG TTA, TTG, CTT, CTC, CTA CTG (t) 3589~591 GGC TGT, TGC 4 319~321 CCC ATG 4 688~690 GCG GAA GAG (u) 4235~237 CAC AAT, AAC 4 688~690 GCG GAA, GAG 4 691~693 GCC GTT, GTC, GTA,GTG (v) 3 274~276 GAC GAA, GAG 4 69~71 GTC ATT, ATC, ATA 4 676~678 GTCATT, ATC, ATA (w) 3 589~591 GGC TGT, TGC 4 69~71 GTC ATT, ATC, ATA 4319~321 CCC ATG 4 688~690 GCG GAA, GAG (x) 4 69~71 GTC ATT, ATC, ATA 4235~237 CAC AAT, AAC 4 688~690 GCG GAA, GAG 4 691~693 GCC GTT, GTC, GTA,GTG

Furthermore, substitutions of the amino acid illustrated in Table IIImay be realized by base substitution, as shown in Table V.

TABLE V Sequence Before ID No. No. Substitution After Substitution (aa)3 106~108 ACG ATG 3 376~378 TTC TAT, TAC (ab) 3 442~444 GGC GAU, GAC 3610~612 GTC CGT, CGC, CGA, CGG, AGA, AGG (ac) 4 151~153 TTC GTT, GTC,GTA, GTG 4 322~324 GAG GAT, GAC (ad) 4 352~354 TTC GTT, GTC, GTA, GTG 4598~600 GCC GAA, GAG (ae) 4 478~480 CGG TGG 4 556~558 CTG CGT, CGC, CGA,CGG, AGA, AGG (af) 3 16~18 CTG ACT, ACC, ACA, ACG 3 106~108 ACG ATG 3376~378 TTC TAT, TAC (ag) 3 55~57 GCG GTT, GTC, GTA, GTG 3 211~213 CGTCAT, CAC 3 376~378 TTC TAT, TAC (ah) 3 106~108 ACG ATG 3 442~444 GGCGAT, GAC 3 610~612 GTC CGT, CGC, CGA, CGG, AGA, AGG (ai) 4 28~30 ACCGAT, GAC 4 352~354 TTC GTT, GTC, GTA, GTG 4 598~600 GCC GAA, GAG (aj) 4109~111 TTC TTA, TTG, CTT, CTC, CTA, CTG 4 322~324 GAG GAT, GAC 4598~600 GCC GAA, GAG (ak) 4 109~111 TTC GTT, GTC, GTA, GTG 4 322~324 GAGGAT, GAC 4 598~600 GCC GAA, GAG

TABLE V-1 (al) 4 121~123 TTC ATT, ATC, ATA 4 151~153 TTC GTT, GTC, GTA,GTG 4 322~324 GAG GAT, GAC (am) 4 136~138 ATG AAA, AAG 4 322~324 GAGCGT, CGC, CGA, CGG, AGA, AGG 4 634~636 TCC TAT, TAC (an) 4 142~144 CTGGTT, GTC, GTA, GTG 4 322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4 634~636TCC TAT, TAC (ao) 4 379~381 CTG TCT, TCC, TCA, TCG, AGT, AGC 4 478~480CGG TGG 4 556~558 CTG CGT, CGC, CGA, CGG, AGA, AGG (ap) 3 16~18 CTG ACT,ACC, ACA, ACG 3 55~57 GCG GTT, GTC, GTA, GTG 3 376~378 TTC TAT, TAC 4136~138 ATG AAA, AAG 4 322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4634~636 TCC TAT, TAC (aq) 3 16~18 CTG ACT, ACC, ACA, ACG 3 55~57 GCGGTT, GTC, GTA, GTG 3 376~378 TTC TAT, TAC 4 142~144 CTG GTT, GTC, GTA,GTG 4 322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4 634~636 TCC TAT, TAC(ar) 3 16~18 CTG GOT, GCC, GCA, GCG 3 55~57 GCG GTT, GTC, GTA, GTG 3376~378 TTC TAT, TAC 4 379~381 CTG TCT, TCC, TCA, TCG, AGU, AGC 4478~480 CGG TGG 4 556~558 CTG CGT, CGC, CGA, CGG, AGA, AGG

TABLE V-2 (as) 3 16~18 CTG ACT, ACC, ACA, ACG 3 106~108 ACG ATG 3376~378 TTC TAT, TAC 4 28~30 ACC GAT, GAC 4 352~354 TTC GTT, GTC, GTA,GTG 4 598~600 GCC GAA, GAG (at) 3 55~57 GCG GTT, GTC, GTA, GTG 3 211~213CGT CAT, CAC 3 376~378 TTC TAT, TAC 4 109~111 TTC TTA, TTG, CTT, CTC,CTA, CTG 4 322~324 GAG GAT, GAC 4 598~600 GCC GAA, GAG (au) 3 55~57 GCGGTT, GTC, GTA, GTG 3 211~213 CGT CAT, CAC 3 376~378 TTC TAT, TAC 4109~111 TTC GTT, GTC, GTA, GTG 4 322~324 GAG GAT, GAC 4 598~600 GCC GAA,GAG (av) 3 106~108 ACG ATG 3 442~444 GGC GAT, GAC 3 610~612 GTC CGT,CGC, CGA, CGG, AGA, AGG 4 121~123 TTC ATT, ATC, ATA 4 151~153 TTC GTT,GTC, GTA, GTG 4 322~324 GAG GAT, GAC (aw) 3 442~444 GGC GAT, GAC 3610~612 GTC CGT, CGC, CGA, CGG, AGA, AGG 4 322~324 GAG GAT, GAC 4598~600 GCC GAA, GAG (ax) 3 106~108 ACG GGT, GGC, GGA, GGG 3 562~564 ACCGGT, GGC, GGA, GGG

TABLE V-3 (ay) 3 106~108 ACG GCT, GCC, GCA, GCG 3 142~144 AAC CAA, CAG(az) 3 142~144 AAC GAA, GAG 4 436~438 CGG GGT, GGC, GGA, GGG (ba) 3106~108 ACG TGG 4 526~528 TAC TGT, TGC (bb) 4 526~528 TAC ATG 4 649~651GAC GGT, GGC, GGA, GGG (bc) 3 106~108 ACG TCT, TCC, TCA, TCG, AGT, AGC 497~99 GCG GTT, GTC, GTA, GTG (bd) 4 526~528 TAC GCT, GCC, GCA, GCG 4649~651 GAC GTT, GTC, GTA, GTG (be) 4 118~120 ACG GTT, GTC, GTA, GTG 4652~654 TGC ATG (bf) 4 97~99 GCG ATG 4 526~528 TAC ACT, ACC, ACA, ACG(bg) 4 118~120 ACG TTA, TTG, CTT, CTC, CTA, CTG 4 649~651 GAC TTA, TTG,CTT, CTC, CTA, CTG (bh) 4 118~120 ACG ATT, ATC, ATA 4 181~183 GCC GTT,GTC, GTA, GTG (bi) 4 181~183 GCC ACT, ACC, ACA, ACG 4 652~654 TGC TCT,TCC, TCA, TCG, AGT, AGC (bj) 4 334~336 AAG GTT, GTC, GTA, GTG 4 649~651GAC ATG (bk) 4 181~183 GCC TGG 4 649~651 GAC CAT, CAC (bl) 4 181~183 GCCTTA, TTG, CTT, CTC, CTA, CTG 4 334~336 AAG ATT, ATC, ATA

TABLE V-4 (bm) 4 436~438 CGG GGT, GGC, GGA, GGG 4 649~651 GAC TCT, TCC,TCA, TCG, AGT, AGC (bn) 4 511~513 AAG GCT, GCC, GCA, GCG 4 649~651 GACACT, ACC, ACA, ACG (bo) 4 448~450 GCG TCT, TCC, TCA, TCG, AGT, AGC 4649~651 GAC TGT, TGC (bp) 4 181~183 GCC GGT, GGC, GGA, GGG 4 448~450 GCGAAT, AAC (bq) 4 181~183 GCC TCT, TCC, TCA, TCG, AGT, AGC 4 478~480 CGGATG (br) 4 478~480 CGG TGT, TGC 4 502~504 ACG GAA, GAG

The plasmid can have, in addition to a gene encoding the α-subunit ofthe nitrile hydratase variant, a gene encoding the β-subunit or anitrile hydratase variant gene or nitryl hydratase variant gene, aconstitution which enables the production of a nitrile hydratase by atransformant or a transformant strain obtained by transforming anarbitrary host cell, such as the regulatory region necessary for theexpression of each gene, the region necessary for autonomous replicationor the like. The arbitrary host cell as used herein may be exemplifiedby Escherichia coli.

The regulatory region necessary for expression may include a promotersequence (including the transcription-regulating operator sequence), aribosome binding sequence (SD sequence), a transcription-terminatingsequence and the like. Specific examples of the promoter sequence mayinclude a trp promoter of tryptophan operon and a lac promoter oflactose operon that are derived from Escherichia coli, and a PL promoterand a PR promoter that are derived from lambda phage. Further,artificially designed or improved sequences such as a tac promoter or atrc promoter may also be used.

The ribosome binding sequence is preferably a sequence having TAAGGAGGTcontained in SEQ ID No: 7. The sequence order of these regulatoryregions on a plasmid is preferably such that the promoter sequence andthe ribosome binding sequence are located upstream to the 5′-terminalthan the gene encoding the nitrile hydratase variant, and thetranscription-terminating sequence is preferably located downstream tothe 3′-terminal than the gene encoding the nitrile hydratase variant.Also, the α-subunit gene and the β-subunit gene of the nitrile hydratasevariant may be expressed as individual independent cistrons by means ofsuch regulatory regions, or may be expressed as a polycistron by meansof a common regulatory region.

Examples of the plasmid vector satisfying the above requirements mayinclude pBR322, pUC18, pBluescript, pKK223-3 and pSC101, which have aregion capable of autonomous replication in Escherichia coli.

For a method of constructing the plasmid of the present invention byinserting the gene encoding the nitrile hydratase variant of the presentinvention into such a plasmid vector, together with those regionsnecessary for expression of the activity of the nitrile hydratasevariant, a method of transforming the plasmid to a desired host cell anda method of producing nitrile hydratase in the transformant, there maybe used those general methods and host cells known in the art ofmolecular biology, biological engineering and genetic engineering asdescribed in, for example, “Molecular Cloning, 3rd Edition” (J. Sambrooket al., Cold Spring Harbor Laboratory Press, 2001) or the like.

The transformant obtained by transforming the above plasmid to a desiredhost cell is cultivated in a culture medium, whereby the nitrilehydratase variant can be produced based on the nitrile hydratase genecarried by the plasmid. When the host cell is Escherichia coli, LBmedium, M9 medium or the like is generally used as the culture mediumfor cultivating the transformant. More preferably, these mediumcomponents may comprise Fe ions and Co ions in an amount of 0.1 μg/mL ormore, or the transformant may be inoculated and then cultivated at asuitable cultivating temperature (in general, from 20 to 50 degreescentigrade).

When the nitrile hydratase variant having the desired enzyme activity toexpress the gene encoding the nitrile hydratase variant of the presentinvention is produced, a gene encoding a protein involved in theactivation of nitrile hydratase may be required in some cases.

A protein involved in the activation of nitrile hydratase is a proteinhaving the property such that the presence or absence of the expressionof the protein directly controls the activation of nitrile hydratase,and it can be exemplified by the protein involved in the activation ofPseudonocardia thermophila-derived nitrile hydratase (nitrilehydratase-activating protein) as described in Japanese Patent Laid-openNo. H11 (1999)-253168. The sequence of the nitrile hydratase-activatingprotein is presented in the Sequence Listing: 5 and 6.

The amide compound can be produced in the following manner using thenitrile hydratase variant of the present invention. First, thetransformant or transformant strain to produce the nitrile hydratasevariant of the present invention is caltivated, and a given cell or agiven product obtained by processing the cells is brought into contactwith a nitrile compound in a solvent. In this manner, a correspondingamide compound is produced.

The term “product obtained by processing the cells” mentioned hereinrefers to an extract or a disruption product of the transformant, apost-separation product such as a crude enzyme preparation obtained byisolating the nitrile hydratase activated fraction from such extract ordisruption product, an enzyme purification product obtained by furtherpurification or the like, and an immobilization product in which thetransformant, or an extract, a disruption product or a post-separationproduct of the transformant is immobilized by using suitable means. Thecontact temperature is not particularly limited, but it is preferably inthe range of not deactivating the nitrile hydratase variant, and morepreferably from 0 to 60 degrees centigrade. As the nitrile compound,there is no particular limitation as long as it is a compound which canact as the substrate for the nitrile hydratase variant of the presentinvention, and it can be preferably exemplified by nitrile compoundshaving 2 to 4 carbon atoms, such as acetonitrile, propionitrile,acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile,crotononitrile, α-hydroxyisobutyronitrile and the like. Theconcentration of the nitrile compound in the aqueous medium is notparticularly limited. The reaction temperature is not particularlylimited, but it is preferably in the range of not deactivating thenitrile hydratase, and more preferably from 0 to 60 degrees centigrade.Furthermore, in order to produce an amide compound with a smaller amountof the enzyme, it is preferable to use a nitrile hydratase varianthaving a certain level of stability under conditions of producing anamide compound.

Subsequently, the operational effect of the present invention will bedescribed in detail. The present inventors have repeatedly conducted anextensive study and as a result, have found a nitrile hydratase variantin which both physical properties relating to the reaction itself andthe enzyme stability are improved as compared to the conventionalnitrile hydratase, comprising substitution of at least one amino acidwith another amino acid to improve two or more properties of nitrilehydratase by substitution of one or more and three or less amino acids.In particular, they have found that with respect to the nitrilehydratase comprising the α-subunit defined in SEQ ID No: 1 in theSequence Listing and the β-subunit defined in SEQ ID No: 2 in theSequence Listing, at least one amino acid is substituted with anotheramino acid, selected from substitution sites of the amino acidconsisting of the above (a) to (l), whereby enzyme stability as well asthe initial reaction rate of the nitrile hydratase can be improved atthe same time. In this way, both of efficiency of the enzymatic reactionand handling of the enzyme can be achieved. Also, by use of the nitrilehydratase in which both of the initial reaction rate and enzymestability are enhanced, the activity value in a unit weight of theenzyme preparation can be increased, and at the same time the risk ofenzyme deactivation due to temperature variation or the like for theindustrial use can be reduced. Accordingly, the amide compound can bestably produced with a smaller amount of the enzyme so that theproduction costs for producing the amide compound can be reduced.

As described above, embodiments of the present invention has beendescribed, but the embodiments described in the present invention areillustrative only, and various other constructions may also be adopted.

EXAMPLES

The present invention is now illustrated in detail below with referenceto the following Examples. However, the present invention is notrestricted to these Examples.

Example 1 Construction of Plasmid (1) Expressing Nitrile Hydratase withModified Ribosome Binding Sequence

A gene fragment of about 0.7 kbp was obtained by the PCR reaction usinga plasmid pPT-DB1 described in Example 3 of Patent Document 1 as thetemplate and the primers defined in SEQ ID Nos: 7 and 8 in the SequenceListing. The above-mentioned PCR fragment was cleaved by means ofrestriction endonucleases EcoRI and NotI, and then this mixture treatedwith restriction endonucleases was subjected to phenol/chloroformextraction and ethanol precipitation to purify the DNA fragment. In thesame manner, pPT-DB1 was cleaved by means of EcoRI and NotI, and thensubjected to agarose gel electrophoresis, through which only the DNAfragment of about 3.9 kbp was cut out of the agarose gel. The thusobtained DNA fragments of about 0.7 kbp and of about 3.9 kbp weresubjected to DNA ligation using a DNA ligation kit (manufactured byTakara Shuzo Co., Ltd.) to prepare a plasmid (1) expressing theabove-mentioned nitrile hydratase with the modified ribosome bindingsequence.

A competent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (1).Moreover, the plasmid was prepared from the above microbial cells by thealkaline SDS extraction method, and the base sequence of the nitrilehydratase gene was determined using a DNA sequencer. Then, it wasconfirmed that the transformant (1) had the modified ribosome bindingsequence in pPT-DB1 as shown in Table 1.

In the production of an amide compound using the thus obtainedtransformant (1) and a transformant MT-10822 containing pPT-DB1(deposited with the International Patent Organism Depositary at theNational Institute of Advanced Industrial Science and Technology, 1-1-1Higashi, Tsukuba-shi, Ibaraki-ken, Japan, under the deposit number FERMBP-5785 from Feb. 7, 1996) to be its base, the initial reaction rateswere compared in the following method.

Comparison of Initial Reaction Rate

5 mL of a liquid LB medium containing 40 μg/mL of ferric sulfateheptahydrate and 10 μg/mL of cobalt chloride dihydrate was prepared in atest tube, and sterilized by autoclaving at 121 degrees centigrade for20 minutes. Ampicillin was added to this medium to have a finalconcentration of 100 μg/mL. Then, on the medium, one platinum loop ofrespective transformants was inoculated and cultivated therein at 37degrees centigrade for about 20 hours with stirring at 200 rpm. 40 μL ofthe resulting culture was taken and suspended in 740 μL of a 54 mMTris-HCl aqueous solution (pH 8.0). To this, 20 μL of acrylonitrile wasadded, and this mixture was gently stirred at 20 degrees centigrade for15 minutes to react, whereby acrylamide was produced. After completionof the reaction, the content of acrylamide in the reaction solution wasanalyzed through HPLC.

Comparison of Thermal Stability of Enzyme

Respective transformants were separated from the resulting culture ofthe above-mentioned transformants by centrifugation (5,000 G×15minutes).

0.1 g of the thus isolated transformants were respectively suspended in20 ml of a 50 mM Tris-HCl aqueous solution (pH 8.0), and heated at 60degrees centigrade for 2 hours. The temperature was returned to 20degrees centigrade after heating, and 0.5 ml of acrylonitrile was addedthereto as the substrate. The reaction was carried out at 20 degreescentigrade for 15 minutes to measure the initial reaction rate.

Analytical Conditions

Analytical Equipment: HPLC manufactured by JASCO Corporation

Column: YMC Pack ODS-A (150×6.00 mm)

Analytical Temperature: 40 degrees centigrade

Mobile Phase: 3% acetonitrile, 10 mM phosphoric acid

Respective transformants were subjected to the reaction and analysisthree times or more to correct variations in the data by means of adispensing operation or the like.

As a result of comparison of the initial reaction rate and thermalstability of the transformant (1) and MT-10822, that is, the amount ofproduced acrylamide under the above reaction conditions, improvement ofthe initial reaction rate by 1.15 times was observed and thermalstability was maintained with the new addition of the modified ribosomebinding sequence shown in Table 1.

TABLE 14 Table 1 Effect on Effect on Improvement Improvement of Reactionof Thermal Rate by Stability by Ribosome RibosomeChange in Base Sequence Binding Binding Transformant Mutated BeforeAfter Sequence Sequence No. Site Substitution Substitution SubstitutionSubstitution 1 Ribosome TGAGAGGAG TAAGGAGGT 1.15 times 1.00 timesBinding Sequence

Reference Example 1 Construction of a Transformant (2) Substituted AminoAcid Having Nitrile Hydratase Activity

In order to obtain a transformant (2) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (av) amino acidsubstitution sites as shown in Table 2, the plasmid described in Example79 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified in the method described in Example 1 toprepare a plasmid (2) encoding the above-mentioned nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (2).

Reference Example 2 Construction of a Transformant (3) Substituted AminoAcid Having Nitrile Hydratase Activity

In order to obtain a transformant (3) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bc) amino acidsubstitution sites as shown in Table 2, introduction of site-specificmutation was performed using a “LA PCR in vitro mutagenesis Kit”manufactured by Takara Shuzo Co., Ltd. (hereinafter referred to as themutagenesis kit). The plasmid (1) expressing nitrile hydratase with themodified ribosome binding sequence described in Example 1 was used asthe template to carry out the PCR reaction.

For the PCR reaction No. 1, a reaction system of 50 μL in totalcontaining 50 pmols of the primer having the sequence defined in SEQ IDNo: 9 in the Sequence Listing and 50 pmols of an M13 primer M4 (havingthe sequence defined in SEQ ID No: 10 in the Sequence Listing) (for thecomposition of the system, the instructions described in the mutagenesiskit were followed) was used, and the reaction consisted of 25 PCRcycles, in which one PCR cycle comprised thermal denaturation (98degrees centigrade) for 15 seconds, annealing (55 degrees centigrade)for 30 seconds and chain extension (72 degrees centigrade) for 120seconds.

For the PCR reaction No. 2, a reaction system of 50 μL in totalcontaining 50 pmols of an MUT4 primer (having the sequence defined inSEQ ID No: 11 in the Sequence Listing) and an M13 primer RV (having thesequence defined in SEQ ID No: 12 in the Sequence Listing) (for thecomposition of the system, the instructions described in the mutagenesiskit were followed) was used, and the reaction was carried out followingthe same procedure as the PCR reaction No. 1.

After completion of the PCR reaction Nos. 1 and 2, 5 μL of the reactionmixture was subjected to agarose gel electrophoresis (where the agaroseconcentration was 1.0 weight %), and an analysis of the DNAamplification product was carried out. As a result, the presence of theamplified DNA product was confirmed. From each of these PCR reactionmixtures, the excess primers and dNTP were removed using Microcon 100(manufactured by Takara Shuzo Co., Ltd.), and then TE was added to eachof the mixtures to prepare 50 μL each of TE solutions. An annealingsolution of 47.5 μL in total containing 0.5 μL of both of the above TEsolutions (for the composition of the system, the instructions describedin the mutagenesis kit were followed) was prepared, and this solutionwas subjected to annealing by performing thermal denaturation of thesolution at 98 degrees centigrade for 10 minutes, subsequently coolingthe solution to 37 degrees centigrade at a constant cooling rate over aperiod of 60 minutes, and then maintaining it at 37 degrees centigradefor 15 minutes. To the thus annealed solution, 0.5 μL of TaKaRa LA Taq(manufactured by Takara Bio Inc.) was added, and the solution was heatedat 72 degrees centigrade for 3 minutes, thus completing the formation ofheterologous double-stranded DNA.

To this was added 50 pmols of an M13 primer M4 (having the sequencedefined in SEQ ID No: 10 in the Sequence Listing) and 50 pmols of an M13primer RV (having the sequence defined in SEQ ID No: 12 in the SequenceListing) to give a reaction system of 50 μL in total, and the reactionconsisted of 25 PCR cycles, in which one PCR cycle comprised thermaldenaturation (98 degrees centigrade) for 15 seconds, annealing (55degrees centigrade) for 30 seconds and chain extension (72 degreescentigrade) for 120 seconds to carry out the PCR reaction No. 3. Aftercompletion of the PCR reaction No. 3, 5 μL of the reaction mixture wassubjected to agarose gel electrophoresis (using Type VIIlow-melting-point agarose, a product by Sigma Corporation; agaroseconcentration of 0.8 weight %), and an analysis of the DNA amplificationproduct was carried out. As a result, the presence of the amplified DNAproduct of about 2 kb was confirmed.

Subsequently, an agarose fragment comprising only the DNA fragment ofabout 2 kb was cut out of the agarose gel. The thus cut agarose fragment(about 0.1 g) was finely pulverized, suspended in 1 ml of a TE solution,and then kept at 55 degrees centigrade for 1 hour, whereby the agarosefragment was completely melted. The resulting agarose melt was thensubjected to phenol/chloroform extraction and ethanol precipitation topurify the DNA fragment. The thus purified DNA fragment was finallydissolved in 10 μL of TE. The amplified DNA fragment of about 2 kb thuspurified was cleaved by means of restriction endonucleases EcoRI andHindIII, and this mixture treated with restriction endonucleases wasthen subjected to phenol/chloroform extraction and ethanol precipitationto purify the DNA fragment. The thus purified DNA fragment was finallydissolved in 10 μL of TE.

Likewise, the plasmid (1) expressing nitrile hydratase with the modifiedribosome binding sequence described in Example 1 was cleaved by means ofEcoRI and HindIII, and then subjected to agarose gel electrophoresis(using Type VII low-melting-point agarose, a product by SigmaCorporation; agarose concentration of 0.7%). An agarose fragmentcomprising only the DNA fragment of about 2.7 kb was cut out of theagarose gel. The thus cut agarose fragment (about 0.1 g) was finelypulverized, suspended in 1 ml of the TE solution, and then kept at 55degrees centigrade for 1 hour, whereby the agarose fragment wascompletely melted. The resulting agarose melt was then subjected tophenol/chloroform extraction and ethanol precipitation to purify the DNAfragment. The thus purified DNA fragment was finally dissolved in 10 μLof TE.

The thus obtained DNA fragments of about 2 kbp and of about 2.7 kbp weresubjected to DNA ligation, using a DNA ligation kit (manufactured byTakara Shuzo Co., Ltd.). Then, a competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed. The aboveoperation was carried out using the plasmid extracted from thetransformant as the template, and using the primer having the sequencedefined in SEQ ID No: 13 instead of the primer defined in SEQ ID No: 9,whereby a plasmid (3) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (3).

Reference Example 3 Construction of a Transformant (4) Substituted AminoAcid Having Nitrile Hydratase Activity

In order to obtain a transformant (4) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bh) amino acidsubstitution sites as shown in Table 2, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 14 and 15 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (4) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (4).

TABLE 2 Change in Amino Acid Change in Trans- Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 2 α-36th Thr Met ACG ATG α-148thGly Asp GGC GAC α-204th Val Arg GTC CGC β-41st Phe Ile TTC ATC β-51stPhe Val TTC GTC β-108th Glu Asp GAG GAT 3 α-36th Thr Ser ACG TCG β-33rdAla Val GCG GTG 4 β-40th Thr Ile ACG ATT β-61st Ala Val GCC GTC

Example 2 Construction of a Transformant (5) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (5) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (a) and (av) aminoacid substitution sites as shown in Table 3, the plasmid (2) recoveredfrom the transformant (2) described in the above Reference Example 1 wasused as the template, and the primer having the sequence defined in SEQID No: 16 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (5) encoding the above nitrile hydratase variant was prepared. Acompetent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (5).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (5) had sequences according tothe purpose in which mutation of 92nd Asp in the α-subunit with Glu wasnewly added to the plasmid (2) of Reference Example 1.

In the production of an amide compound using the thus obtainedtransformant (5) and the transformant (2) to be its base, the initialreaction rate and thermal stability were compared in the same manner asin Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu was newly added to the transformant (5), so that the initialreaction rate was improved by 1.65 times and thermal stability wasimproved by 1.25 times, as compared to those of the transformant (2).

Example 3 Construction of a Transformant (6) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (6) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (a) and (bc) aminoacid substitution sites as shown in Table 3, the plasmid (3) recoveredfrom the transformant (3) described in the above Reference Example 2 wasused as the template, and the primer having the sequence defined in SEQID No: 16 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (6) encoding the above nitrile hydratase variant was prepared. Acompetent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (6).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (6) had sequences according tothe purpose in which mutation of 92nd Asp in the α-subunit with Glu wasnewly added to the plasmid (3) of Reference Example 2. In the productionof an amide compound using the thus obtained transformant (6) and thetransformant (3) to be its base, the initial reaction rate and thermalstability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu was newly added to the transformant (6), so that the initialreaction rate was improved by 1.63 times and thermal stability wasimproved by 1.23 times, as compared to those of the transformant (3).

Example 4 Construction of a Transformant (7) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (7) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (a) and (bh) aminoacid substitution sites as shown in Table 3, the plasmid (4) recoveredfrom the transformant (4) described in the above Reference Example 3 wasused as the template, and the primer having the sequence defined in SEQID No: 16 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (7) encoding the above nitrile hydratase variant was prepared. Acompetent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (7).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (7) had sequences according tothe purpose in which mutation of 92nd Asp in the α-subunit with Glu wasnewly added to the plasmid (4) of Reference Example 3. In the productionof an amide compound using the thus obtained transformant (7) and thetransformant (4) to be its base, the initial reaction rate and thermalstability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu was newly added to the transformant (7), so that the initialreaction rate was improved by 1.58 times and thermal stability wasimproved by 1.30 times, as compared to those of the transformant (4).

TABLE 3 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- α-92nd α-92nd No. Site tion tion tion tionSubstitution Substitution 5 α-36th Thr Met ACG ATG 1.65 times 1.25 timesα-92nd Asp Glu GAC GAG α-148th Gly Asp GGC GAC α-204th Val Arg GTC CGCβ-41st Phe Ile TTC ATC β-51st Phe Val TTC GTC β-108th Glu Asp GAG GAT 6α-36th Thr Ser ACG TCG 1.63 times 1.23 times α-92nd Asp Glu GAC GAGβ-33rd Ala Val GCG GTG 7 α-92nd Asp Glu GAC GAG 1.58 times 1.30 timesβ-40th Thr Ile ACG ATT β-61st Ala Val GCC GTC

Reference Example 4 Construction of a Transformant (8) Substituted AminoAcid Having Nitrile Hydratase Activity

In order to obtain a transformant (8) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ak) amino acidsubstitution sites as shown in Table 4, the plasma described in Example68 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (8) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (8).

Reference Example 5 Construction of a Transformant (9) Substituted AminoAcid Having Nitrile Hydratase Activity

In order to obtain a transformant (9) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ap) amino acidsubstitution sites as shown in Table 4, the plasma described in Example73 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (9) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (9).

Reference Example 6 Construction of a Transformant (10) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (10) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bp) amino acidsubstitution sites as shown in Table 4, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 17 and 18 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (10) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (10).

TABLE 4 Change in Amino Acid Change in Trans- Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 8 β-37th Phe Val TTC GTC β-108thGlu Asp GAG GAT β-200th Ala Glu GCC GAG 9 α-6th Leu Thr CTG ACG α-19thAla Val GCG GTG α-126th Phe Tyr TTC TAC β-46th Met Lys ATG AAG β-108thGlu Arg GAG CGG β-212th Ser Tyr TCC TAC 10 β-61st Ala Gly GCC GGCβ-150th Ala Asn GCG AAT

Example 5 Construction of a Transformant (11) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (11) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (b) and (ak) aminoacid substitution sites as shown in Table 5, the plasmid (8) recoveredfrom the transformant (8) described in the above Reference Example 4 wasused as the template, and the primer having the sequence defined in SEQID No: 19 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (11) encoding the above nitrile hydratase variant was prepared.A competent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (11).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (11) had sequences according tothe purpose in which mutation of 94th Met in the α-subunit with Ile wasnewly added to the plasmid (8) of Reference Example 4. In the productionof an amide compound using the thus obtained transformant (11) and thetransformant (8) to be its base, the initial reaction rate and thermalstability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 94th Met in the α-subunitwith Ile was newly added to the transformant (11), so that the initialreaction rate was improved by 1.45 times and thermal stability wasimproved by 1.38 times, as compared to those of the transformant (8).

Example 6 Construction of a Transformant (12) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (12) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (b) and (ap) aminoacid substitution sites as shown in Table 5, the plasmid (9) recoveredfrom the transformant (9) described in the above Reference Example 5 wasused as the template, and the primer having the sequence defined in SEQID No: 19 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (12) encoding the above nitrile hydratase variant was prepared.A competent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (12).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (12) had sequences according tothe purpose in which mutation of 94th Met in the α-subunit with Ile wasnewly added to the plasmid (9) of Reference Example 5. In the productionof an amide compound using the thus obtained transformant (12) and thetransformant (9) to be its base, the initial reaction rate and thermalstability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 94th Met in the α-subunitwith Ile was newly added to the transformant (12), so that the initialreaction rate was improved by 1.40 times and thermal stability wasimproved by 1.25 times, as compared to those of the transformant (9).

Example 7 Construction of a Transformant (13) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (13) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (b) and (bp) aminoacid substitution sites as shown in Table 5, the plasmid (10) recoveredfrom the transformant (10) described in the above Reference Example 6was used as the template, and the primer having the sequence defined inSEQ ID No: 19 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (13) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (13). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (13) hadsequences according to the purpose in which mutation of 94th Met in theα-subunit with Ile was newly added to the plasmid (10) of ReferenceExample 6. In the production of an amide compound using the thusobtained transformant (13) and the transformant (10) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 94th Met in the α-subunitwith Ile was newly added to the transformant (13), so that the initialreaction rate was improved by 1.32 times and thermal stability wasimproved by 1.35 times, as compared to those of the transformant (10).

TABLE 5 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- α-94th α-94th No. Site tion tion tion tionSubstitution Substitution 11 α-94th Met Ile ATG ATC 1.45 times 1.38times β-37th Phe Val TTC GTC β-108th Glu Asp GAG GAT β-200th Ala Glu GCCGAG 12 α-6th Leu Thr CTG ACG 1.40 times 1.25 times α-19th Ala Val GCGGTG α-94th Met Ile ATG ATC α-126th Phe Tyr TTC TAC β-46th Met Lys ATGAAG β-108th Glu Arg GAG CGG β-212th Ser Tyr TCC TAC 13 α-94th Met IleATG ATC 1.32 times 1.35 times β-61st Ala Gly GCC GGC β-150th Ala Asn GCGAAT

Reference Example 7 Construction of a Transformant (14) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (14) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (an) amino acidsubstitution sites as shown in Table 6, the plasma described in Example71 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (14) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (14).

Reference Example 8 Construction of a Transformant (15) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (15) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (be) amino acidsubstitution sites as shown in Table 6, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 20 and 21 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (15) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (15).

Reference Example 9 Construction of a Transformant (16) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (16) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (br) amino acidsubstitution sites as shown in Table 6, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 22 and 23 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (16) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (16).

TABLE 6 Change in Amino Acid Change in Trans- Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 14 β-48th Leu Val CTG GTG β-108thGlu Arg GAG CGG β-212th Ser Tyr TCC TAC 15 β-40th Thr Val ACG GTGβ-218th Cys Met TGC ATG 16 β-160th Arg Cys CGG TGT β-168th Thr Glu ACGGAG

Example 8 Construction of a Transformant (17) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (17) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (c) and (an) aminoacid substitution sites as shown in Table 7, the plasmid (14) recoveredfrom the transformant (14) described in the above Reference Example 7was used as the template, and the primer having the sequence defined inSEQ ID No: 24 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (17) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (17). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (17) hadsequences according to the purpose in which mutation of 197th Gly in theα-subunit with Cys was newly added to the plasmid (14) of ReferenceExample 7. In the production of an amide compound using the thusobtained transformant (17) and the transformant (14) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys was newly added to the transformant (17), so that the initialreaction rate was improved by 1.80 times and thermal stability wasimproved by 1.25 times, as compared to those of the transformant (14).

Example 9 Construction of a Transformant (18) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (18) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (c) and (be) aminoacid substitution sites as shown in Table 7, the plasmid (15) recoveredfrom the transformant (15) described in the above Reference Example 8was used as the template, and the primer having the sequence defined inSEQ ID No: 24 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (18) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (18). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (18) hadsequences according to the purpose in which mutation of 197th Gly in theα-subunit with Cys was newly added to the plasmid (15) of ReferenceExample 8. In the production of an amide compound using the thusobtained transformant (18) and the transformant (15) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys was newly added to the transformant (18), so that the initialreaction rate was improved by 1.86 times and thermal stability wasimproved by 1.40 times, as compared to those of the transformant (15).

Example 10 Construction of a Transformant (19) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (19) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (c) and (br) aminoacid substitution sites as shown in Table 7, the plasmid (16) recoveredfrom the transformant (16) described in the above Reference Example 9was used as the template, and the primer having the sequence defined inSEQ ID No: 24 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (19) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (19). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (19) hadsequences according to the purpose in which mutation of 197th Gly in theα-subunit with Cys was newly added to the plasmid (16) of ReferenceExample 9. In the production of an amide compound using the thusobtained transformant (19) and the transformant (16) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys was newly added to the transformant (19), so that the initialreaction rate was improved by 1.68 times and thermal stability wasimproved by 1.20 times, as compared to those of the transformant (16).

TABLE 7 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- α-197th α-197th No. Site tion tion tion tionSubstitution Substitution 17 α-197th Gly Cys GGC TGC 1.80 times 1.25times β-48th Leu Val CTG GTG β-108th Glu Arg GAG CGG β-212th Ser Tyr TCCTAC 18 α-197th Gly Cys GGC TGC 1.86 times 1.40 times β-40th Thr Val ACGGTG β-218th Cys Met TGC ATG 19 α-197th Gly Cys GGC TGC 1.68 times 1.20times β-160th Arg Cys CGG TGT β-168th Thr Glu ACG GAG

Reference Example 10 Construction of a Transformant (20) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (20) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ar) amino acidsubstitution sites as shown in Table 8, the plasma described in Example75 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (20) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (20).

Reference Example 11 Construction of a Transformant (21) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (21) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ax) amino acidsubstitution sites as shown in Table 8, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 25 and 26 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (21) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (21).

Reference Example 12 Construction of a Transformant (22) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (22) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bd) amino acidsubstitution sites as shown in Table 8, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 27 and 28 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (22) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (22).

TABLE 8 Change in Amino Acid Change in Trans- Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 20 α-6th Leu Ala CTG GCG α-19thAla Val GCG GTG α-126th Phe Tyr TTC TAC β-127th Leu Ser CTG TCG β-160thArg Trp CGG TGG β-186th Leu Arg CTG CGG 21 α-36th Thr Gly ACG GGGα-188th Thr Gly ACC GGC 22 β-176th Tyr Ala TAC GCC β-217th Asp Val GACGTC

Example 11 Construction of a Transformant (23) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (23) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (d) and (ar) aminoacid substitution sites as shown in Table 9, the plasmid (20) recoveredfrom the transformant (20) described in the above Reference Example 10was used as the template, and the primer having the sequence defined inSEQ ID No: 29 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (23) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (23). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (23) hadsequences according to the purpose in which mutation of 4th Val in theβ-subunit with Met was newly added to the plasmid (20) of ReferenceExample 10. In the production of an amide compound using the thusobtained transformant (23) and the transformant (20) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 4th Val in the β-subunit withMet was newly added to the transformant (23), so that the initialreaction rate was improved by 1.25 times and thermal stability wasimproved by 1.35 times, as compared to those of the transformant (20).

Example 12 Construction of a Transformant (24) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (24) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (d) and (ax) aminoacid substitution sites as shown in Table 9, the plasmid (21) recoveredfrom the transformant (21) described in the above Reference Example 11was used as the template, and the primer having the sequence defined inSEQ ID No: 29 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (24) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (24). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (24) hadsequences according to the purpose in which mutation of 4th Val in theβ-subunit with Met was newly added to the plasmid (21) of ReferenceExample 11. In the production of an amide compound using the thusobtained transformant (24) and the transformant (21) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 4th Val in the β-subunit withMet was newly added to the transformant (24), so that the initialreaction rate was improved by 1.32 times and thermal stability wasimproved by 1.39 times, as compared to those of the transformant (21).

Example 13 Construction of a Transformant (25) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (25) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (d) and (bd) aminoacid substitution sites as shown in Table 9, the plasmid (22) recoveredfrom the transformant (22) described in the above Reference Example 12was used as the template, and the primer having the sequence defined inSEQ ID No: 29 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (25) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (19).

Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (25) had sequences according tothe purpose in which mutation of 4th Val in the β-subunit with Met wasnewly added to the plasmid (22) of Reference Example 12. In theproduction of an amide compound using the thus obtained transformant(25) and the transformant (22) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result of comparison of the initial reaction rate and thermalstability between the transformant (25) and the transformant (22), itwas found that mutation of 4th Val in the β-subunit with Met was newlyadded to the transformant (25), so that the initial reaction rate wasimproved by 1.25 times and thermal stability was improved by 1.25 times,as compared to those of the transformant (22).

TABLE 9 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-4th β-4th No. Site tion tion tion tionSubstitution Substitution 23 α-6th Leu Ala CTG GCG 1.25 times 1.35 timesα-19th Ala Val GCG GTG α-126th Phe Tyr TTC TAC β-4th Val Met GTG ATGβ-127th Leu Ser CTG TCG β-160th Arg Trp CGG TGG β-186th Leu Arg CTG CGG24 α-36th Thr Gly ACG GGG 1.32 times 1.39 times α-188th Thr Gly ACC GGCβ-4th Val Met GTG ATG 25 β-4th Val Met GTG ATG 1.25 times 1.25 timesβ-176th Tyr Ala TAC GCC β-217th Asp Val GAC GTC

Reference Example 13 Construction of a Transformant (26) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (26) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ao) amino acidsubstitution sites as shown in Table 10, the plasma described in Example72 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (26) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (26).

Reference Example 14 Construction of a Transformant (27) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (27) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (at) amino acidsubstitution sites as shown in Table 10, the plasma described in Example77 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (27) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (27).

TABLE 10 Change in Amino Acid Change in Trans- Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 26 β-127th Leu Ser CTG TCGβ-160th Arg Trp CGG TGG β-186th Leu Arg CTG CGG 27 α-19th Ala Val GCGGTG α-71st Arg His CGT CAT α-126th Phe Tyr TTC TAC β-37th Phe Leu TTCCTC β-108th Glu Asp GAG GAT β-200th Ala Glu GCC GAG

Comparative Example 1 Construction of a Transformant (28) SubstitutedAmino Acid Having Improved Nitrile Hydratase Activity

In order to obtain a transformant (28) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at 8th of the β-subunitand (ao) amino acid substitution sites as shown in Table 11, the plasmid(26) recovered from the transformant (26) described in the aboveReference Example 13 was used as the template, and the primer having thesequence defined in SEQ ID No: 30 in the Sequence Listing was used forcarrying out the method using the mutagenesis kit described in ReferenceExample 2, whereby a plasmid (28) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (28). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (28) hadsequences according to the purpose in which mutation of 8th Gly in theβ-subunit with Ala was newly added to the plasmid (26) of ReferenceExample 13. In the production of an amide compound using the thusobtained transformant (28) and the transformant (26) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result of comparison, it was found that mutation of 8th Gly in theβ-subunit with Ala was newly added to the transformant (28), so that theinitial reaction rate was improved by 1.35 times and thermal stabilitywas lowered by 0.65 times, as compared to those of the transformant(26).

Comparative Example 2 Construction of a Transformant (29) SubstitutedAmino Acid Having Improved Nitrile Hydratase Activity

In order to obtain a transformant (29) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at 8th of the β-subunitand (at) amino acid substitution sites as shown in Table 11, the plasmid(27) recovered from the transformant (27) described in the aboveReference Example 14 was used as the template, and the primer having thesequence defined in SEQ ID No: 30 in the Sequence Listing was used forcarrying out the method using the mutagenesis kit described in ReferenceExample 2, whereby a plasmid (29) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (29). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (29) hadsequences according to the purpose in which mutation of 8th Gly in theβ-subunit with Ala was newly added to the plasmid (27) of ReferenceExample 14. In the production of an amide compound using the thusobtained transformant (29) and the transformant (27) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 8th Gly in the β-subunit withAla was newly added to the transformant (29), so that the initialreaction rate was improved by 1.40 times and thermal stability waslowered by 0.31 times, as compared to those of the transformant (27).

Comparative Example 3 Construction of a Transformant (30) SubstitutedAmino Acid Having Improved Nitrile Hydratase Activity

In order to obtain a transformant (30) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at 8th of the β-subunitand (br) amino acid substitution sites as shown in Table 11, the plasmid(16) recovered from the transformant (16) described in the aboveReference Example 9 was used as the template, and the primer having thesequence defined in SEQ ID No: 30 in the Sequence Listing was used forcarrying out the method using the mutagenesis kit described in ReferenceExample 2, whereby a plasmid (30) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (30). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (30) hadsequences according to the purpose in which mutation of 8th Gly in theβ-subunit with Ala was newly added to the plasmid (16) of ReferenceExample 9. In the production of an amide compound using the thusobtained transformant (30) and the transformant (16) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 8th Gly in the β-subunit withAla was newly added to the transformant (30), so that the initialreaction rate was improved by 1.32 times and thermal stability waslowered by 0.52 times, as compared to those of the transformant (16).

TABLE 11 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-8th β-8th No. Site tion tion tion tionSubstitution Substitution 28 α-8th Gly Ala GGC GCC 1.35 times 0.65 timesβ-127th Leu Ser CTG TCG β-160h Arg Trp CGG TGG β-186th Leu Arg CTG CGG29 α-19th Ala Val GCG GTG 1.40 times 0.31 times α-71st Arg His CGT CATα-126th Phe Tyr TTC TAC β-8th Gly Ala GGC GCC β-37th Phe Leu TTC CTCβ-108th Glu Asp GAG GAT β-200th Ala Glu GCC GAG 30 β-8th Gly Ala GGC GCC1.32 times 0.52 times β-160th Arg Cys CGG TGT β-168th Thr Glu ACG GAG

Reference Example 15 Construction of a Transformant (31) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (31) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (au) amino acidsubstitution sites as shown in Table 12, the plasma described in Example78 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (31) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (31).

Reference Example 16 Construction of a Transformant (32) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (32) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bf) amino acidsubstitution sites as shown in Table 12, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 31 and 32 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (32) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (32).

TABLE 12 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 31 α-19th Ala Val GCG GTG α-71stArg His CGT CAT α-126th Phe Tyr TTC TAC β-37th Phe Val TTC GTC β-108thGlu Asp GAG GAT β-200th Ala Glu GCC GAG 32 β-33rd Ala Met GCG ATGβ-176th Tyr Thr TAC ACC

Example 14 Construction of a Transformant (33) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (33) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (f) and (au) aminoacid substitution sites as shown in Table 13, the plasmid (31) recoveredfrom the transformant (31) described in the above Reference Example 15was used as the template, and the primer having the sequence defined inSEQ ID No: 33 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (33) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (33). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (33) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn was newly added to the plasmid (31) of ReferenceExample 15. In the production of an amide compound using the thusobtained transformant (33) and the transformant (31) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn was newly added to the transformant (33), so that the initialreaction rate was improved by 1.29 times and thermal stability wasimproved by 1.82 times, as compared to those of the transformant (31).

Example 15 Construction of a Transformant (34) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (34) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (f) and (bf) aminoacid substitution sites as shown in Table 13, the plasmid (32) recoveredfrom the transformant (32) described in the above Reference Example 16was used as the template, and the primer having the sequence defined inSEQ ID No: 33 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (34) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (34). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (34) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn was newly added to the plasmid (32) of ReferenceExample 16. In the production of an amide compound using the thusobtained transformant (34) and the transformant (32) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn was newly added to the transformant (34), so that the initialreaction rate was improved by 1.25 times and thermal stability wasimproved by 1.76 times, as compared to those of the transformant (32).

Example 16 Construction of a Transformant (35) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (35) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (f) and (bp) aminoacid substitution sites as shown in Table 13, the plasmid (10) recoveredfrom the transformant (10) described in the above Reference Example 6was used as the template, and the primer having the sequence defined inSEQ ID No: 33 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (35) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (35). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (35) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn was newly added to the plasmid (10) of ReferenceExample 6. In the production of an amide compound using the thusobtained transformant (35) and the transformant (10) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn was newly added to the transformant (35), so that the initialreaction rate was improved by 1.30 times and thermal stability wasimproved by 1.72 times, as compared to those of the transformant (10).

TABLE 13 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-79th β-79th No. Site tion tion tion tionSubstitution Substitution 33 α-19th Ala Val GCG GTG 1.29 times 1.82times α-71st Arg His CGT CAT α-126th Phe Tyr TTC TAC β-37th Phe Val TTCGTC β-79th His Asn CAC AAC β-108h Glu Asp GAG GAT β-200th Ala Glu GCCGAG 34 β-33rd Ala Met GCG ATG 1.25 times 1.76 times β-79th His Asn CACAAC β-176th Tyr Thr TAC ACC 35 β-61st Ala Gly GCC GGC 1.30 times 1.72times β-79th His Asn CAC AAC β-150th Ala Asn GCG AAT

Reference Example 17 Construction of a Transformant (36) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (36) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (aa) amino acidsubstitution sites as shown in Table 14, the plasma described in Example58 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (36) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (36).

Reference Example 18 Construction of a Transformant (37) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (37) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ah) amino acidsubstitution sites as shown in Table 14, the plasma described in Example65 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (37) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (37).

Reference Example 19 Construction of a Transformant (38) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (38) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (aq) amino acidsubstitution sites as shown in Table 14, the plasma described in Example74 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (38) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (38).

TABLE 14 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 36 α-36th Thr Met ACG ATG α-126thPhe Tyr TTC TAC 37 α-36th Thr Met ACG ATG α-148th Gly Asp GGC GACα-204th Val Arg GTC CGC 38 α-6th Leu Thr CTG ACG α-19th Ala Val GCG GTGα-126th Phe Tyr TTC TAC β-48th Leu Val CTG GTG β-108th Glu Arg GAG CGGβ-212th Ser Tyr TCC TAC

Example 17 Construction of a Transformant (39) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (39) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (g) and (aa) aminoacid substitution sites as shown in Table 15, the plasmid (36) recoveredfrom the transformant (36) described in the above Reference Example 17was used as the template, and the primer having the sequence defined inSEQ ID No: 34 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (39) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (39). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (39) hadsequences according to the purpose in which mutation of 96th Gln in theβ-subunit with Arg was newly added to the plasmid (36) of ReferenceExample 17. In the production of an amide compound using the thusobtained transformant (39) and the transformant (36) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 96th Gln in the β-subunitwith Arg was newly added to the transformant (39), so that the initialreaction rate was improved by 1.33 times and thermal stability wasimproved by 1.25 times, as compared to those of the transformant (36).

Example 18 Construction of a Transformant (40) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (40) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (g) and (ah) aminoacid substitution sites as shown in Table 15, the plasmid (37) recoveredfrom the transformant (37) described in the above Reference Example 18was used as the template, and the primer having the sequence defined inSEQ ID No: 34 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (40) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (40). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (40) hadsequences according to the purpose in which mutation of 96th Gln in theβ-subunit with Arg was newly added to the plasmid (37) of ReferenceExample 18. In the production of an amide compound using the thusobtained transformant (40) and the transformant (37) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 96th Gln in the β-subunitwith Arg was newly added to the transformant (40), so that the initialreaction rate was improved by 1.25 times and thermal stability wasimproved by 1.36 times, as compared to those of the transformant (37).

Example 19 Construction of a Transformant (41) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (41) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (g) and (aq) aminoacid substitution sites as shown in Table 15, the plasmid (38) recoveredfrom the transformant (38) described in the above Reference Example 19was used as the template, and the primer having the sequence defined inSEQ ID No: 34 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (41) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (41). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (41) hadsequences according to the purpose in which mutation of 96th Gln in theβ-subunit with Arg was newly added to the plasmid (38) of ReferenceExample 19. In the production of an amide compound using the thusobtained transformant (41) and the transformant (38) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 96th Gln in the β-subunitwith Arg was newly added to the transformant (41), so that the initialreaction rate was improved by 1.35 times and thermal stability wasimproved by 1.42 times, as compared to those of the transformant (38).

TABLE 15 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-96th β-96th No. Site tion tion tion tionSubstitution Substitution 39 α-36th Thr Met ACG ATG 1.33 times 1.25times α-126th Phe Tyr TTC TAC β-96th Gln Arg CAG CGT 40 α-36th Thr MetACG ATG 1.25 times 1.36 times α-148th Gly Asp GGC GAC α-204th Val ArgGTC CGC β-96th Gln Arg CAG CGT 41 α-6th Leu Thr CTG ACG 1.35 times 1.42times α-19th Ala Val GCG GTG α-126th Phe Tyr TTC TAC β-48th Leu Val CTGGTG β-96th Gln Arg CAG CGT β-108th Glu Arg GAG CGG β-212th Ser Tyr TCCTAC

Reference Example 20 Construction of a Transformant (42) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (42) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ae) amino acidsubstitution sites as shown in Table 16, the plasma described in Example62 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (42) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (42).

Reference Example 21 Construction of a Transformant (43) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (43) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bk) amino acidsubstitution sites as shown in Table 16, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 35 and 36 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (43) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (43).

TABLE 16 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 42 β-160th Arg Trp CGG TGGβ-186th Leu Arg CTG CGG 43 β-61st Ala Trp GCC TGG β-217th Asp His GACCAC

Example 20 Construction of a Transformant (44) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (44) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (h) and (ae) aminoacid substitution sites as shown in Table 17, the plasmid (42) recoveredfrom the transformant (42) described in the above Reference Example 20was used as the template, and the primer having the sequence defined inSEQ ID No: 37 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (44) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (44). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (44) hadsequences according to the purpose in which mutation of 107th Pro in theβ-subunit with Met was newly added to the plasmid (42) of ReferenceExample 20. In the production of an amide compound using the thusobtained transformant (44) and the transformant (42) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 107th Pro in the β-subunitwith Met was newly added to the transformant (44), so that the initialreaction rate was improved by 1.34 times and thermal stability wasimproved by 2.25 times, as compared to those of the transformant (42).

Example 21 Construction (45) of a Transformant (45) Substituted AminoAcid Having Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (45) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (h) and (au) aminoacid substitution sites as shown in Table 17, the plasmid (31) recoveredfrom the transformant (31) described in the above Reference Example 15was used as the template, and the primer having the sequence defined inSEQ ID No: 68 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (45) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (45). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (45) hadsequences according to the purpose in which mutation of 107th Pro in theβ-subunit with Met was newly added to the plasmid (31) of ReferenceExample 15. In the production of an amide compound using the thusobtained transformant (45) and the transformant (31) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 107th Pro in the β-subunitwith Met was newly added to the transformant (45), so that the initialreaction rate was improved by 1.40 times and thermal stability wasimproved by 2.12 times, as compared to those of the transformant (31).

Example 22 Construction of a Transformant (46) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (46) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (h) and (bk) aminoacid substitution sites as shown in Table 17, the plasmid (43) recoveredfrom the transformant (43) described in the above Reference Example 21was used as the template, and the primer having the sequence defined inSEQ ID No: 37 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (46) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (46). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (46) hadsequences according to the purpose in which mutation of 107th Pro in theβ-subunit with Met was newly added to the plasmid (43) of ReferenceExample 21. In the production of an amide compound using the thusobtained transformant (46) and the transformant (43) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 107th Pro in the β-subunitwith Met was newly added to the transformant (46), so that the initialreaction rate was improved by 1.32 times and thermal stability wasimproved by 2.40 times, as compared to those of the transformant (43).

TABLE 17 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-107th β-107th No. Site tion tion tion tionSubstitution Substitution 44 β-107th Pro Met CCC ATG 1.34 times 2.25times β-160th Arg Trp CGG TGG β-186th Leu Arg CTG CGG 45 α-19th Ala ValGCG GTG 1.40 times 2.12 times α-71st Arg His CGT CAT α-126th Phe Tyr TTCTAC β-37th Phe Val TTC GTC β-107th Pro Met CCC ATG β-108th Glu Asp GAGGAT β-200th Ala Glu GCC GAG 46 β-61st Ala Trp GCC TGG 1.32 times 2.40times β-107th Pro Met CCC ATG β-217th Asp His GAC CAC

Example 23 Construction of a Transformant (47) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (47) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (i) and (aa) aminoacid substitution sites as shown in Table 18, the plasmid (36) recoveredfrom the transformant (36) described in the above Reference Example 17was used as the template, and the primer having the sequence defined inSEQ ID No: 38 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (47) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (47). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (47) hadsequences according to the purpose in which mutation of 226th Val in theβ-subunit with Ile was newly added to the plasmid (36) of ReferenceExample 17. In the production of an amide compound using the thusobtained transformant (47) and the transformant (36) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 226th Val in the β-subunitwith Ile was newly added to the transformant (47), so that the initialreaction rate was improved by 1.26 times and thermal stability wasimproved by 1.29 times, as compared to those of the transformant (36).

Example 24 Construction (48) of a Transformant (48) Substituted AminoAcid Having Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (48) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (i) and (ak) aminoacid substitution sites as shown in Table 18, the plasmid (8) recoveredfrom the transformant (8) described in the above Reference Example 4 wasused as the template, and the primer having the sequence defined in SEQID No: 38 in the Sequence Listing was used for carrying out the methodusing the mutagenesis kit described in Reference Example 2, whereby aplasmid (48) encoding the above nitrile hydratase variant was prepared.A competent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (48).Moreover, the plasmid was prepared from the above-mentioned microbialcells by the alkaline SDS extraction method, and the base sequence ofthe nitrile hydratase gene was determined using a DNA sequencer. Then,it was confirmed that the transformant (48) had sequences according tothe purpose in which mutation of 226th Val in the β-subunit with Ile wasnewly added to the plasmid (8) of Reference Example 4. In the productionof an amide compound using the thus obtained transformant (48) and thetransformant (8) to be its base, the initial reaction rate and thermalstability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 226th Val in the β-subunitwith Ile was newly added to the transformant (48), so that the initialreaction rate was improved by 1.35 times and thermal stability wasimproved by 1.27 times, as compared to those of the transformant (8).

Example 25 Construction of a Transformant (49) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (49) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (i) and (be) aminoacid substitution sites as shown in Table 18, the plasmid (15) recoveredfrom the transformant (15) described in the above Reference Example 8was used as the template, and the primer having the sequence defined inSEQ ID No: 38 in the Sequence Listing was used for carrying out themethod using the mutagenesis kit described in Reference Example 2,whereby a plasmid (49) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (49). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (49) hadsequences according to the purpose in which mutation of 226th Val in theβ-subunit with Ile was newly added to the plasmid (15) of ReferenceExample 8. In the production of an amide compound using the thusobtained transformant (49) and the transformant (15) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 226th Val in the β-subunitwith Ile was newly added to the transformant (49), so that the initialreaction rate was improved by 1.25 times and thermal stability wasimproved by 1.30 times, as compared to those of the transformant (15).

TABLE 18 Change in Amino Acid Change in Base Improvement of SequenceSequence Improvement of Thermal Trans- Before After Before AfterReaction Rate by Stability by formant Mutated Substitu- Substitu-Substitu- Substitu- β-226th β-226th No. Site tion tion tion tionSubstitution Substitution 47 α-36th Thr Met ACG ATG 1.26 times 1.29times α-126th Phe Tyr TTC TAC β-226th Val Ile GTC ATC 48 β-37th Phe ValTTC GTC 1.35 times 1.27 times β-108th Glu Asp GAG GAT β-200th Ala GluGCC GAG β-226th Val Ile GTC ATC 49 β-40th Thr Val ACG GTG 1.25 times1.30 times β-218th Cys Met TGC ATG β-226th Val Ile GTC ATC

Reference Example 22 Construction of a Transformant (50) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (50) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (af) amino acidsubstitution sites as shown in Table 19, the plasma described in Example63 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (50) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (50).

Reference Example 23 Construction of a Transformant (51) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a plasmid encoding the nitrile hydratase variantobtained by mutating nitrile hydratase at (bq) amino acid substitutionsites as shown in Table 19, introduction of site-specific mutation wasperformed using the mutagenesis kit described in the above ReferenceExample 2. The plasmid (1) expressing nitrile hydratase with themodified ribosome binding sequence described in Example 1 was used asthe template, the primers having the sequence defined in SEQ ID Nos: 39and 40 in the Sequence Listing were used for repeatedly carrying out themethod described in Reference Example 2 per mutation point, whereby aplasmid (51) encoding the above nitrile hydratase variant was prepared.A competent cell of Escherichia coli HB101 (manufactured by Toyobo Co.,Ltd.) was transformed with the plasmid to obtain a transformant (51).

Reference Example 24 Construction of a Transformant (52) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (52) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bj) amino acidsubstitution sites as shown in Table 19, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 41 and 42 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (52) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (52).

TABLE 19 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 50 α-6th Leu Thr CTG ACG α-36thThr Met ACG ATG α-126th Phe Tyr TTC TAC 51 β-61st Ala Ser GCC TCGβ-160th Arg Met CGG ATG 52 β-112th Lys Val AAG GTG β-217th Asp Met GACATG

Example 26 Construction of a Transformant (53) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (53) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m−1) and (af) aminoacid substitution sites as shown in Table 20, the plasmid (50) recoveredfrom the transformant (50) described in the above Reference Example 22was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (53) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (53). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (53) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (50) of Reference Example 22. In theproduction of an amide compound using the thus obtained transformant(53) and the transformant (50) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (53), so that the initial reaction rate wasimproved by 1.67 times and thermal stability was improved by 1.45 times,as compared to those of the transformant (50).

Example 27 Construction of a Transformant (54) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (54) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m−1) and (bq) aminoacid substitution sites as shown in Table 20, the plasmid (51) recoveredfrom the transformant (51) described in the above Reference Example 23was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (54) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (54). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (54) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (51) of Reference Example 23. In theproduction of an amide compound using the thus obtained transformant(54) and the transformant (51) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (54), so that the initial reaction rate wasimproved by 1.59 times and thermal stability was improved by 1.32 times,as compared to those of the transformant (51).

Example 28 Construction of a Transformant (55) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (55) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m−1) and (bj) aminoacid substitution sites as shown in Table 20, the plasmid (52) recoveredfrom the transformant (52) described in the above Reference Example 24was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (55) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (55). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (55) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (52) of Reference Example 24. In theproduction of an amide compound using the thus obtained transformant(55) and the transformant (52) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (55), so that the initial reaction rate wasimproved by 1.62 times and thermal stability was improved by 1.26 times,as compared to those of the transformant (52).

TABLE 20 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-13th and α-13th and formant Mutated Substitu-Substitu- Substitu- Substitu- α-94th α-94th No. Site tion tion tion tionSubstitution Substitution 53 α-6th Leu Thr CTG ACG 1.67 times 1.45 timesα-13th Ile Leu ATC CTC α-36th Thr Met ACG ATG α-94th Met Ile ATG ATCα-126th Phe Tyr TTC TAC 54 α-13th Ile Leu ATC CTC 1.59 times 1.32 timesα-94th Met Ile ATG ATC β-61st Ala Ser GCC TCG β-160th Arg Met CGG ATG 55α-13th Ile Leu ATC CTC 1.62 times 1.26 times α-94th Met Ile ATG ATCβ-112th Lys Val AAG GTG β-217th Asp Met GAC ATG

Reference Example 25 Construction of a Transformant (56) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (56) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (am) amino acidsubstitution sites as shown in Table 21, the plasma described in Example70 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (56) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (56).

Reference Example 26 Construction of a Transformant (57) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (57) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ay) amino acidsubstitution sites as shown in Table 21, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 44 and 45 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (57) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (57).

TABLE 21 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 56 β-46th Met Lys ATG AAG β-108thGlu Arg GAG CGG β-212th Ser Tyr TCC TAC 57 α-36th Thr Ala ACG GCG α-48thAsn Gln AAC CAA

Example 29 Construction of a Transformant (58) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (58) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m-2) and (am) aminoacid substitution sites as shown in Table 22, the plasmid (56) recoveredfrom the transformant (56) described in the above Reference Example 25was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 34 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (58) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (58). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (56) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 96th Gln in the β-subunit with Argwere newly added to the plasmid (56) of Reference Example 25. In theproduction of an amide compound using the thus obtained transformant(58) and the transformant (56) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 96th Gln in the β-subunit with Arg were newlyadded to the transformant (58), so that the initial reaction rate wasimproved by 1.53 times and thermal stability was improved by 1.32 times,as compared to those of the transformant (56).

Example 30 Construction of a Transformant (59) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (59) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m-2) and (at) aminoacid substitution sites as shown in Table 22, the plasmid (27) recoveredfrom the transformant (27) described in the above Reference Example 14was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 34 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (59) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (59). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (59) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 96th Gln in the β-subunit with Argwere newly added to the plasmid (27) of Reference Example 14. In theproduction of an amide compound using the thus obtained transformant(59) and the transformant (27) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 96th Gln in the β-subunit with Arg were newlyadded to the transformant (59), so that the initial reaction rate wasimproved by 1.49 times and thermal stability was improved by 1.28 times,as compared to those of the transformant (27).

Example 31 Construction of a Transformant (60) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (60) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (m-2) and (ay) aminoacid substitution sites as shown in Table 22, the plasmid (57) recoveredfrom the transformant (57) described in the above Reference Example 26was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43 and 34 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (60) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (60). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (60) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu and mutation of 96th Gln in the β-subunit with Argwere newly added to the plasmid (57) of Reference Example 26. In theproduction of an amide compound using the thus obtained transformant(60) and the transformant (57) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu and mutation of 96th Gln in the β-subunit with Arg were newlyadded to the transformant (60), so that the initial reaction rate wasimproved by 1.39 times and thermal stability was improved by 1.45 times,as compared to those of the transformant (57).

TABLE 22 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-13th and α-13th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-96th β-96th No. Site tion tion tion tionSubstitutions Substitutions 58 α-13th Ile Leu ATC CTC 1.53 times 1.32times β-46th Met Lys ATG AAG β-96th Gln Arg CAG CGT β-108th Glu Arg GAGCGG β-212th Ser Tyr TCC TAC 59 α-13th Ile Leu ATC CTC 1.49 times 1.28times α-19th Ala Val GCG GTG α-71st Arg His CGT CAT α-126th Phe Tyr TTCTAC β-37th Phe Leu TTC CTC β-96th Gln Arg CAG CGT β-108th Glu Asp GAGGAT β-200th Ala Glu GCC GAG 60 α-13th Ile Leu ATC CTC 1.39 times 1.45times α-36th Thr Ala ACG GCG α-48th Asn Gln AAC CAA β-96th Gln Arg CAGCGT

Example 32 Construction of a Transformant (61) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (61) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n−1) and (af) aminoacid substitution sites as shown in Table 23, the plasmid (50) recoveredfrom the transformant (50) described in the above Reference Example 22was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (61) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (61). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (61) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (50) of Reference Example 22. In theproduction of an amide compound using the thus obtained transformant(61) and the transformant (50) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (61), so that the initial reaction rate wasimproved by 1.65 times and thermal stability was improved by 1.36 times,as compared to those of the transformant (50).

Example 33 Construction of a Transformant (62) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (62) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n−1) and (ao) aminoacid substitution sites as shown in Table 23, the plasmid (26) recoveredfrom the transformant (26) described in the above Reference Example 13was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (62) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (62). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (62) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (26) of Reference Example 13. In theproduction of an amide compound using the thus obtained transformant(62) and the transformant (26) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (62), so that the initial reaction rate wasimproved by 1.72 times and thermal stability was improved by 1.47 times,as compared to those of the transformant (26).

Example 34 Construction of a Transformant (63) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (63) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n−1) and (ax) aminoacid substitution sites as shown in Table 23, the plasmid (21) recoveredfrom the transformant (21) described in the above Reference Example 11was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 19 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (63) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (63). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (63) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 94th Met in the α-subunit with Ilewere newly added to the plasmid (21) of Reference Example 11. In theproduction of an amide compound using the thus obtained transformant(63) and the transformant (21) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 94th Met in the α-subunit with Ile were newlyadded to the transformant (63), so that the initial reaction rate wasimproved by 1.55 times and thermal stability was improved by 1.27 times,as compared to those of the transformant (21).

TABLE 23 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-27th and α-27th and formant Mutated Substitu-Substitu- Substitu- Substitu- α-94th α-94th No. Site tion tion tion tionSubstitutions Substitutions 61 α-6th Leu Thr CTG ACG 1.65 times 1.36times α-27th Met Ile ATG ATC α-36th Thr Met ACG ATG α-94th Met Ile ATGATC α-126th Phe Tyr TTC TAC 62 α-27th Met Ile ATG ATC 1.72 times 1.47times α-94th Met Ile ATG ATC β-127th Leu Ser CTG TCG β-160th Arg Trp CGGTGG β-186th Leu Arg CTG CGG 63 α-27th Met Ile ATG ATC 1.55 times 1.27times α-36th Thr Gly ACG GGG α-94th Met Ile ATG ATC α-188th Thr Gly ACCGGC

Reference Example 27 Construction of a Transformant (64) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (64) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (aj) amino acidsubstitution sites as shown in Table 24, the plasma described in Example67 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (64) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (64).

Reference Example 28 Construction of a Transformant (65) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (65) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (as) amino acidsubstitution sites as shown in Table 24, the plasma described in Example76 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (65) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (65).

Reference Example 29 Construction of a Transformant (66) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (66) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bb) amino acidsubstitution sites as shown in Table 24, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 47 and 48 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (66) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (66).

TABLE 24 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 64 β-37th Phe Leu TTC CTC β-108thGlu Asp GAG GAT β-200th Ala Glu GCC GAG 65 α-6th Leu Thr CTG ACG α-36thThr Met ACG ATG α-126th Phe Tyr TTC TAC β-10th Thr Asp ACC GAC β-118thPhe Val TTC GTC β-200th Ala Glu GCC GAG 66 β-176th Tyr Met TAC ATGβ-217th Asp Gly GAC GGC

Example 35 Construction of a Transformant (67) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (67) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n-2) and (aj) aminoacid substitution sites as shown in Table 25, the plasmid (64) recoveredfrom the transformant (64) described in the above Reference Example 27was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 68 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (67) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (67). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (67) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 107th Pro in the β-subunit with Metwere newly added to the plasmid (64) of Reference Example 27. In theproduction of an amide compound using the thus obtained transformant(67) and the transformant (64) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 107th Pro in the β-subunit with Met were newlyadded to the transformant (67), so that the initial reaction rate wasimproved by 1.53 times and thermal stability was improved by 2.23 times,as compared to those of the transformant (64).

Example 36 Construction of a Transformant (68) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (68) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n-2) and (as) aminoacid substitution sites as shown in Table 25, the plasmid (65) recoveredfrom the transformant (65) described in the above Reference Example 28was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 37 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (68) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (68). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (68) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 107th Pro in the β-subunit with Metwere newly added to the plasmid (65) of Reference Example 28. In theproduction of an amide compound using the thus obtained transformant(68) and the transformant (65) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 107th Pro in the β-subunit with Met were newlyadded to the transformant (68), so that the initial reaction rate wasimproved by 1.55 times and thermal stability was improved by 2.15 times,as compared to those of the transformant (65).

Example 37 Construction of a Transformant (69) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (69) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (n-2) and (bb) aminoacid substitution sites as shown in Table 25, the plasmid (66) recoveredfrom the transformant (66) described in the above Reference Example 29was used as the template, and the primers having the sequence defined inSEQ ID Nos: 46 and 37 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (69) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (69). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (69) hadsequences according to the purpose in which mutation of 27th Met in theα-subunit with Ile and mutation of 107th Pro in the β-subunit with Metwere newly added to the plasmid (66) of Reference Example 29. In theproduction of an amide compound using the thus obtained transformant(69) and the transformant (66) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 27th Met in the α-subunitwith Ile and mutation of 107th Pro in the β-subunit with Met were newlyadded to the transformant (69), so that the initial reaction rate wasimproved by 1.46 times and thermal stability was improved by 1.92 times,as compared to those of the transformant (66).

TABLE 25 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-27th and α-27th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-107th β-107th No. Site tion tion tiontion Substitutions Substitutions 67 α-27th Met Ile ATG ATC 1.53 times2.23 times β-37th Phe Leu TTC CTC β-107th Pro Met CCC ATG β-108th GluAsp GAG GAT β-200th Ala Glu GCC GAG 68 α-6th Leu Thr CTG ACG 1.55 times2.15 times α-27th Met Ile ATG ATC α-36th Thr Met ACG ATG α-126th Phe TyrTTC TAC β-10th Thr Asp ACC GAC β-107th Pro Met CCC ATG β-118th Phe ValTTC GTC β-200th Ala Glu GCC GAG 69 α-27th Met Ile ATG ATC 1.46 times1.92 times β-107th Pro Met CCC ATG β-176th Tyr Met TAC ATG β-217th AspGly GAC GGC

Reference Example 30 Construction of a Transformant (70) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (70) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (al) amino acidsubstitution sites as shown in Table 26, the plasma described in Example69 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (70) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (70).

Reference Example 31 Construction of a Transformant (71) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (71) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (aw) amino acidsubstitution sites as shown in Table 26, the plasma described in Example80 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (71) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (71).

Reference Example 32 Construction of a Transformant (72) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (72) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bl) amino acidsubstitution sites as shown in Table 26, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 49 and 50 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (72) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (72).

TABLE 26 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 70 β-41st Phe Ile TTC ATC β-51stPhe Val TTC GTC β-108th Glu Asp GAG GAT 71 α-148th Gly Asp GGC GACα-204th Val Arg GTC CGC β-108th Glu Asp GAG GAT β-200th Ala Glu GCC GAG72 β-61st Ala Leu GCC CTC β-112th Lys Ile AAG ATT

Example 38 Construction of a Transformant (73) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (73) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (q) and (al) aminoacid substitution sites as shown in Table 27, the plasmid (70) recoveredfrom the transformant (70) described in the above Reference Example 30was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16 and 38 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (73) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (73). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (73) hadsequences according to the purpose in which mutation of 92nd Asp in theα-subunit with Glu and mutation of 226th Val in the β-subunit with Ilewere newly added to the plasmid (70) of Reference Example 30. In theproduction of an amide compound using the thus obtained transformant(73) and the transformant (70) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu and mutation of 226th Val in the β-subunit with Ile were newlyadded to the transformant (73), so that the initial reaction rate wasimproved by 2.00 times and thermal stability was improved by 1.52 times,as compared to those of the transformant (70).

Example 39 Construction of a Transformant (74) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (74) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (q) and (aw) aminoacid substitution sites as shown in Table 27, the plasmid (71) recoveredfrom the transformant (71) described in the above Reference Example 31was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16 and 38 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (74) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (74). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (74) hadsequences according to the purpose in which mutation of 92nd Asp in theα-subunit with Glu and mutation of 226th Val in the β-subunit with Ilewere newly added to the plasmid (71) of Reference Example 31. In theproduction of an amide compound using the thus obtained transformant(74) and the transformant (71) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu and mutation of 226th Val in the β-subunit with Ile were newlyadded to the transformant (74), so that the initial reaction rate wasimproved by 1.78 times and thermal stability was improved by 1.44 times,as compared to those of the transformant (71).

Example 40 Construction of a Transformant (75) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (75) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (q) and (bl) aminoacid substitution sites as shown in Table 27, the plasmid (72) recoveredfrom the transformant (72) described in the above Reference Example 32was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16 and 38 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (75) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (75). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (75) hadsequences according to the purpose in which mutation of 92nd Asp in theα-subunit with Glu and mutation of 226th Val in the β-subunit with Ilewere newly added to the plasmid (72) of Reference Example 32. In theproduction of an amide compound using the thus obtained transformant(75) and the transformant (72) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu and mutation of 226th Val in the β-subunit with Ile were newlyadded to the transformant (75), so that the initial reaction rate wasimproved by 1.85 times and thermal stability was improved by 1.38 times,as compared to those of the transformant (72).

TABLE 27 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-92nd and α-92nd and formant Mutated Substitu-Substitu- Substitu- Substitu- β-226th β-226th No. Site tion tion tiontion Substitution Substitution 73 α-92nd Asp Glu GAC GAG 2.00 times 1.52times β-41st Phe Ile TTC ATC β-51st Phe Val TTC GTC β-108th Glu Asp GAGGAT β-226th Val Ile GTC ATC 74 α-92nd Asp Glu GAC GAG 1.78 times 1.44times α-148th Gly Asp GGC GAC α-204th Val Arg GTC CGC β-108th Glu AspGAG GAT β-200th Ala Glu GCC GAG β-226th Val Ile GTC ATC 75 α-92nd AspGlu GAC GAG 1.85 times 1.38 times β-61st Ala Leu GCC CTC β-112th Lys IleAAG ATT β-226th Val Ile GTC ATC

Reference Example 33 Construction of a Transformant (76) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (76) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bi) amino acidsubstitution sites as shown in Table 28, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 51 and 52 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (76) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (76).

Reference Example 34 Construction of a Transformant (77) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (77) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bm) amino acidsubstitution sites as shown in Table 28, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 53 and 54 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (77) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (77).

TABLE 28 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 76 β-61st Ala Thr GCC ACG β-218thCys Ser TGC TCC 77 β-146th Arg Gly CGG GGG β-217th Asp Ser GAC AGC

Example 41 Construction of a Transformant (78) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (78) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (o) and (an) aminoacid substitution sites as shown in Table 29, the plasmid (14) recoveredfrom the transformant (14) described in the above Reference Example 7was used as the template, and the primers having the sequence defined inSEQ ID Nos: 29 and 33 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (78) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (78). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (78) hadsequences according to the purpose in which mutation of 4th Val in theβ-subunit with Met and mutation of 79th His in the β-subunit with Asnwere newly added to the plasmid (14) of Reference Example 7. In theproduction of an amide compound using the thus obtained transformant(78) and the transformant (14) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 4th Val in the β-subunit withMet and mutation of 79th His in the β-subunit with Asn were newly addedto the transformant (78), so that the initial reaction rate was improvedby 1.60 times and thermal stability was improved by 1.46 times, ascompared to those of the transformant (14).

Example 42 Construction of a Transformant (79) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (79) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (o) and (bi) aminoacid substitution sites as shown in Table 29, the plasmid (76) recoveredfrom the transformant (76) described in the above Reference Example 33was used as the template, and the primers having the sequence defined inSEQ ID Nos: 29 and 33 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (79) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (79). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (79) hadsequences according to the purpose in which mutation of 4th Val in theβ-subunit with Met and mutation of 79th His in the β-subunit with Asnwere newly added to the plasmid (76) of Reference Example 33. In theproduction of an amide compound using the thus obtained transformant(79) and the transformant (76) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 4th Val in the β-subunit withMet and mutation of 79th His in the β-subunit with Asn were newly addedto the transformant (79), so that the initial reaction rate was improvedby 1.38 times and thermal stability was improved by 1.35 times, ascompared to those of the transformant (76).

Example 43 Construction of a Transformant (80) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (80) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (o) and (bm) aminoacid substitution sites as shown in Table 29, the plasmid (77) recoveredfrom the transformant (77) described in the above Reference Example 34was used as the template, and the primers having the sequence defined inSEQ ID Nos: 29 and 33 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (80) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (80). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (80) hadsequences according to the purpose in which mutation of 4th Val in theβ-subunit with Met and mutation of 79th His in the β-subunit with Asnwere newly added to the plasmid (77) of Reference Example 34. In theproduction of an amide compound using the thus obtained transformant(80) and the transformant (77) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 4th Val in the β-subunit withMet and mutation of 79th His in the β-subunit with Asn were newly addedto the transformant (80), so that the initial reaction rate was improvedby 1.52 times and thermal stability was improved by 1.28 times, ascompared to those of the transformant (77).

TABLE 29 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After β-4th and β-4th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-79th β-79th No. Site tion tion tion tionSubstitution Substitution 78 β-4th Val Met GTG ATG 1.60 times 1.46 timesβ-48th Leu Val CTG GTG β-79th His Asn CAC AAC β-108th Glu Arg GAG CGGβ-212th Ser Tyr TCC TAC 79 β-4th Val Met GTG ATG 1.38 times 1.35 timesβ-61st Ala Thr GCC ACG β-79th His Asn CAC AAC β-218th Cys Ser TGC TCC 80β-4th Val Met GTG ATG 1.52 times 1.28 times β-79th His Asn CAC AACβ-146th Arg Gly CGG GGG β-217th Asp Ser GAC AGC

Reference Example 35 Construction of a Transformant (81) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (81) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ag) amino acidsubstitution sites as shown in Table 30, the plasma described in Example64 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (81) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (81).

Reference Example 36 Construction of a Transformant (82) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (82) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ai) amino acidsubstitution sites as shown in Table 30, the plasma described in Example66 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (82) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (82).

TABLE 30 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 81 α-19th Ala Val GCG GTG α-71stArg His CGT CAT α-126th Phe Tyr TTC TAC 82 β-10th Thr Asp ACC GACβ-118th Phe Val TTC GTC β-200th Ala Glu GCC GAG

Example 44 Construction of a Transformant (83) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (83) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (p) and (ag) aminoacid substitution sites as shown in Table 31, the plasmid (81) recoveredfrom the transformant (81) described in the above Reference Example 35was used as the template, and the primers having the sequence defined inSEQ ID Nos: 33 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (83) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (83). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (83) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (81) of Reference Example 35. In theproduction of an amide compound using the thus obtained transformant(83) and the transformant (81) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (83), so that the initial reaction rate wasimproved by 1.25 times and thermal stability was improved by 2.16 times,as compared to those of the transformant (81).

Example 45 Construction of a Transformant (84) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (84) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (p) and (ai) aminoacid substitution sites as shown in Table 31, the plasmid (82) recoveredfrom the transformant (82) described in the above Reference Example 36was used as the template, and the primers having the sequence defined inSEQ ID Nos: 33 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (84) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (84). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (84) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (82) of Reference Example 36. In theproduction of an amide compound using the thus obtained transformant(84) and the transformant (82) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (84), so that the initial reaction rate wasimproved by 1.27 times and thermal stability was improved by 2.10 times,as compared to those of the transformant (82).

Example 46 Construction (85) of a Transformant (85) Substituted AminoAcid Having Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (85) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (p) and (aq) aminoacid substitution sites as shown in Table 31, the plasmid (38) recoveredfrom the transformant (38) described in the above Reference Example 19was used as the template, and the primers having the sequence defined inSEQ ID Nos: 33 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (85) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (85). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (85) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (38) of Reference Example 31. In theproduction of an amide compound using the thus obtained transformant(85) and the transformant (38) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (85), so that the initial reaction rate wasimproved by 1.33 times and thermal stability was improved by 2.52 times,as compared to those of the transformant (38).

TABLE 31 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After β-79th and β-79th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-230th β-230th No. Site tion tion tiontion Substitution Substitution 83 α-19th Ala Val GCG GTG 1.25 times 2.16times α-71st Arg His CGT CAT α-126th Phe Tyr TTC TAC β-79th His Asn CACAAC β-230th Ala Glu GCG GAG 84 β-10th Thr Asp ACC GAC 1.27 times 2.10times β-79th His Asn CAC AAC β-118th Phe Val TTC GTC β-200th Ala Glu GCCGAG β-230th Ala Glu GCG GAG 85 α-6th Leu Thr CTG ACG 1.33 times 2.52times α-19th Ala Val GCG GTG α-126th Phe Tyr TTC TAC β-48th Leu Val CTGGTG β-79th His Asn CAC AAC β-108th Glu Arg GAG CGG β-212th Ser Tyr TCCTAC β-230th Ala Glu GCG GAG

Reference Example 37 Construction of a Transformant (86) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (86) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ad) amino acidsubstitution sites as shown in Table 32, the plasma described in Example61 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (86) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (86).

Reference Example 38 Construction of a Transformant (87) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (87) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bg) amino acidsubstitution sites as shown in Table 32, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 55 and 56 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (87) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (87).

TABLE 32 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 86 β-118th Phe Val TTC GTCβ-200th Ala Glu GCC GAG 87 β-40th Thr Leu ACG CTG β-217th Asp Leu GACCTC

Example 47 Construction of a Transformant (88) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (88) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (j) and (ad) aminoacid substitution sites as shown in Table 33, the plasmid (86) recoveredfrom the transformant (86) described in the above Reference Example 37was used as the template, and the primers having the sequence defined inSEQ ID Nos: 57 and 58 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (88) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (88). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (88) hadsequences according to the purpose in which mutation of 110th Glu in theβ-subunit with Asn and mutation of 231st Ala in the β-subunit with Valwere newly added to the plasmid (86) of Reference Example 37. In theproduction of an amide compound using the thus obtained transformant(88) and the transformant (86) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 110th Glu in the β-subunitwith Asn and mutation of 231st Ala in the β-subunit with Val were newlyadded to the transformant (88), so that the initial reaction rate wasimproved by 1.29 times and thermal stability was improved by 1.62 times,as compared to those of the transformant (86).

Example 48 Construction of a Transformant (89) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (89) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (j) and (aj) aminoacid substitution sites as shown in Table 33, the plasmid (64) recoveredfrom the transformant (64) described in the above Reference Example 27was used as the template, and the primers having the sequence defined inSEQ ID Nos: 57 and 58 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (89) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (89). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (89) hadsequences according to the purpose in which mutation of 110th Glu in theβ-subunit with Asn and mutation of 231st Ala in the β-subunit with Valwere newly added to the plasmid (64) of Reference Example 27. In theproduction of an amide compound using the thus obtained transformant(89) and the transformant (64) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 110th Glu in the β-subunitwith Asn and mutation of 231st Ala in the β-subunit with Val were newlyadded to the transformant (89), so that the initial reaction rate wasimproved by 1.34 times and thermal stability was improved by 1.83 times,as compared to those of the transformant (64).

Example 49 Construction of a Transformant (90) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (90) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (j) and (bg) aminoacid substitution sites as shown in Table 33, the plasmid (87) recoveredfrom the transformant (87) described in the above Reference Example 38was used as the template, and the primers having the sequence defined inSEQ ID Nos: 57 and 58 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (90) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (90). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (90) hadsequences according to the purpose in which mutation of 110th Glu in theβ-subunit with Asn and mutation of 231st Ala in the β-subunit with Valwere newly added to the plasmid (87) of Reference Example 38. In theproduction of an amide compound using the thus obtained transformant(90) and the transformant (87) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 110th Glu in the β-subunitwith Asn and mutation of 231st Ala in the β-subunit with Val were newlyadded to the transformant (90), so that the initial reaction rate wasimproved by 1.25 times and thermal stability was improved by 1.46 times,as compared to those of the transformant (87).

TABLE 33 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After β-110th and β-110th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-231st β-231st No. Site tion tion tiontion Substitution Substitution 88 β-110th Glu Asn GAG AAC 1.29 times1.62 times β-118th Phe Val TTC GTC β-200th Ala Glu GCC GAG β-231st AlaVal GCC GTC 89 β-37th Phe Leu TTC CTC 1.34 times 1.83 times β-108th GluAsp GAG GAT β-110th Glu Asn GAG AAC β-200th Ala Glu GCC GAG β-231st AlaVal GCC GTC 90 β-40th Thr Leu ACG CTG 1.25 times 1.46 times β-110th GluAsn GAG AAC β-217th Asp Leu GAC CTC β-231st Ala Val GCC GTC

Example 50 Construction of a Transformant (91) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (91) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (k) and (ad) aminoacid substitution sites as shown in Table 34, the plasmid (86) recoveredfrom the transformant (86) described in the above Reference Example 37was used as the template, and the primers having the sequence defined inSEQ ID Nos: 59 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (91) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (91). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (91) hadsequences according to the purpose in which mutation of 206th Pro in theβ-subunit with Leu and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (86) of Reference Example 37. In theproduction of an amide compound using the thus obtained transformant(91) and the transformant (86) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 206th Pro in the β-subunitwith Leu and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (91), so that the initial reaction rate wasimproved by 1.44 times and thermal stability was improved by 1.42 times,as compared to those of the transformant (86).

Example 51 Construction of a Transformant (92) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (92) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (k) and (as) aminoacid substitution sites as shown in Table 34, the plasmid (65) recoveredfrom the transformant (65) described in the above Reference Example 28was used as the template, and the primers having the sequence defined inSEQ ID Nos: 59 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (92) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (92). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (92) hadsequences according to the purpose in which mutation of 206th Pro in theβ-subunit with Leu and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (65) of Reference Example 28. In theproduction of an amide compound using the thus obtained transformant(92) and the transformant (65) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 206th Pro in the β-subunitwith Leu and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (92), so that the initial reaction rate wasimproved by 1.48 times and thermal stability was improved by 1.39 times,as compared to those of the transformant (65).

Example 52 Construction of a Transformant (93) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (93) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (k) and (av) aminoacid substitution sites as shown in Table 34, the plasmid (2) recoveredfrom the transformant (2) described in the above Reference Example 1 wasused as the template, and the primers having the sequence defined in SEQID Nos: 59 and 60 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (93) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (93). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (93) hadsequences according to the purpose in which mutation of 206th Pro in theβ-subunit with Leu and mutation of 230th Ala in the β-subunit with Gluwere newly added to the plasmid (2) of Reference Example 1. In theproduction of an amide compound using the thus obtained transformant(93) and the transformant (2) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 206th Pro in the β-subunitwith Leu and mutation of 230th Ala in the β-subunit with Glu were newlyadded to the transformant (93), so that the initial reaction rate wasimproved by 1.36 times and thermal stability was improved by 1.52 times,as compared to those of the transformant (2).

TABLE 34 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After β-206th and β-206th and formant Mutated Substitu-Substitu- Substitu- Substitu- β-230th β-230th No. Site tion tion tiontion Substitution Substitution 91 β-118th Phe Val TCC GTC 1.44 times1.42 times β-200th Ala Glu GCC GAG β-206th Pro Leu CCG CTG β-230th AlaGlu GCG GAG 92 α-6th Leu Thr CTG ACG 1.48 times 1.39 times α-36th ThrMet ACG ATG α-126th Phe Tyr TTC TAC β-10th Thr Asp ACC GAC β-118th PheVal TTC GTC β-200th Ala Glu GCC GAG β-206th Pro Leu CCG CTG β-230th AlaGlu GCG GAG 93 α-36th Thr Met ACG ATG 1.36 times 1.52 times α-148th GlyAsp GGC GAC α-204th Val Arg GTC CGC β-41st Phe Ile TTC ATC β-51st PheVal TTC GTC β-108th Glu Asp GAG GAT β-206th Pro Leu CCG CTG β-230th AlaGlu GCG GAG

Reference Example 39 Construction of a Transformant (94) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (94) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (bo) amino acidsubstitution sites as shown in Table 35, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 61 and 62 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (94) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (94).

TABLE 35 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 94 β-150th Ala Ser GCG TCGβ-217th Asp Cys GAC TGT

Example 53 Construction of a Transformant (95) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (95) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (1) and (ag) aminoacid substitution sites as shown in Table 36, the plasmid (81) recoveredfrom the transformant (81) described in the above Reference Example 35was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43, 46 and 57 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (95) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (95). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (95) had sequences according to the purpose in whichmutation of 13th Ile in the α-subunit with Leu, mutation of 27th Met inthe α-subunit with Ile and mutation of 110th Glu in the β-subunit withAsn were newly added to the plasmid (81) of Reference Example 35. In theproduction of an amide compound using the thus obtained transformant(95) and the transformant (81) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 27th Met in the α-subunit with Ile and mutation of110th Glu in the β-subunit with Asn were newly added to the transformant(95), so that the initial reaction rate was improved by 1.53 times andthermal stability was improved by 1.76 times, as compared to those ofthe transformant (81).

Example 54 Construction of a Transformant (96) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (96) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (1) and (am) aminoacid substitution sites as shown in Table 36, the plasmid (56) recoveredfrom the transformant (56) described in the above Reference Example 25was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43, 46 and 57 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (96) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (96). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (96) had sequences according to the purpose in whichmutation of 13th Ile in the α-subunit with Leu, mutation of 27th Met inthe α-subunit with Ile and mutation of 110th Glu in the β-subunit withAsn were newly added to the plasmid (56) of Reference Example 25. In theproduction of an amide compound using the thus obtained transformant(96) and the transformant (56) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 27th Met in the α-subunit with Ile and mutation of110th Glu in the β-subunit with Asn were newly added to the transformant(96), so that the initial reaction rate was improved by 1.49 times andthermal stability was improved by 1.69 times, as compared to those ofthe transformant (56).

Example 55 Construction of a Transformant (97) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (97) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (1) and (bo) aminoacid substitution sites as shown in Table 36, the plasmid (94) recoveredfrom the transformant (94) described in the above Reference Example 39was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43, 46 and 57 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (97) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (97). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (97) had sequences according to the purpose in whichmutation of 13th Ile in the α-subunit with Leu, mutation of 27th Met inthe α-subunit with Ile and mutation of 110th Glu in the β-subunit withAsn were newly added to the plasmid (94) of Reference Example 39. In theproduction of an amide compound using the thus obtained transformant(97) and the transformant (94) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 27th Met in the α-subunit with Ile and mutation of110th Glu in the β-subunit with Asn were newly added to the transformant(97), so that the initial reaction rate was improved by 1.37 times andthermal stability was improved by 1.83 times, as compared to those ofthe transformant (94).

TABLE 36 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-13th, α-27th α-13th, α-27th formant MutatedSubstitu- Substitu- Substitu- Substitu- and β-110th and β-110th No. Sitetion tion tion tion Substitution Substitution 95 α-13th Ile Leu ATC CTC1.53 times 1.76 times α-19th Ala Val GCG GTG α-27th Met Ile ATG ATCα-71st Arg His CGT CAT α-126th Phe Tyr TTC TAC β-110th Glu Asn GAG AAC96 α-13th Ile Leu ATC CTC 1.49 times 1.69 times α-27th Met Ile ATG ATCβ-46th Met Lys ATG AAG β-108th Glu Arg GAG CGG β-110th Glu Asn GAG AACβ-212th Ser Tyr TCC TAC 97 α-13th Ile Leu ATC CTC 1.37 times 1.83 timesα-27th Met Ile ATG ATC β-110th Glu Asn GAG AAC β-150th Ala Ser GCG TCGβ-217th Asp Cys GAC TGT

Reference Example 40 Construction of a Transformant (98) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (98) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ab) amino acidsubstitution sites as shown in Table 37, the plasma described in Example59 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (98) encoding the above nitrile hydratasevariant. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (98).

TABLE 37 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 98 α-148th Gly Asp GGC GACα-204th Val Arg GTC CGC

Example 56 Construction of a Transformant (99) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (99) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (r) and (ab) aminoacid substitution sites as shown in Table 38, the plasmid (98) recoveredfrom the transformant (98) described in the above Reference Example 40was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43, 59 and 38 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (99) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (99). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (99) had sequences according to the purpose in whichmutation of 13th Ile in the α-subunit with Leu, mutation of 206th Pro inthe β-subunit with Leu and mutation of 226th Val in the β-subunit withIle were newly added to the plasmid (98) of Reference Example 40. In theproduction of an amide compound using the thus obtained transformant(99) and the transformant (98) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 206th Pro in the β-subunit with Leu and mutationof 226th Val in the β-subunit with Ile were newly added to thetransformant (99), so that the initial reaction rate was improved by1.85 times and thermal stability was improved by 1.36 times, as comparedto those of the transformant (98).

Example 57 Construction of a Transformant (100) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (100) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (r) and (ai) aminoacid substitution sites as shown in Table 38, the plasmid (82) recoveredfrom the transformant (82) described in the above Reference Example 36was used as the template, and the primers having the sequence defined inSEQ ID Nos: 43, 59 and 38 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (100) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (100). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (100) had sequences according to the purpose in whichmutation of 13th Ile in the α-subunit with Leu, mutation of 206th Pro inthe β-subunit with Leu and mutation of 226th Val in the β-subunit withIle were newly added to the plasmid (82) of Reference Example 36. In theproduction of an amide compound using the thus obtained transformant(100) and the transformant (82) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 206th Pro in the β-subunit with Leu and mutationof 226th Val in the β-subunit with Ile were newly added to thetransformant (100), so that the initial reaction rate was improved by1.72 times and thermal stability was improved by 1.42 times, as comparedto those of the transformant (82).

Example 58 Construction of a Transformant (101) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (101) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (r) and (bh) aminoacid substitution sites as shown in Table 38, the plasmid (4) recoveredfrom the transformant (4) described in the above Reference Example 3 wasused as the template, and the primers having the sequence defined in SEQID Nos: 43, 59 and 38 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (101) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (101). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (101) hadsequences according to the purpose in which mutation of 13th Ile in theα-subunit with Leu, mutation of 206th Pro in the β-subunit with Leu andmutation of 226th Val in the β-subunit with Ile were newly added to theplasmid (4) of Reference Example 3. In the production of an amidecompound using the thus obtained transformant (101) and the transformant(4) to be its base, the initial reaction rate and thermal stability werecompared in the same manner as in Example 1.

As a result, it was found that mutation of 13th Ile in the α-subunitwith Leu, mutation of 206th Pro in the β-subunit with Leu and mutationof 226th Val in the β-subunit with Ile were newly added to thetransformant (101), so that the initial reaction rate was improved by1.65 times and thermal stability was improved by 1.29 times, as comparedto those of the transformant (4).

TABLE 38 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-13th, β-206th α-13th, β-206th formant MutatedSubstitu- Substitu- Substitu- Substitu- and β-226th and β-226th No. Sitetion tion tion tion Substitution Substitution 99 α-13th Ile Leu ATC CTC1.85 times 1.36 times α-148th Gly Asp GGC GAC α-204th Val Arg GTC CGCβ-206th Pro Leu CCG CTG β-226th Val Ile GTC ATC 100 α-13th Ile Leu ATCCTC 1.72 times 1.42 times β-10th Thr Asp ACC GAC β-118th Phe Val TTC GTCβ-200th Ala Glu GCC GAG β-206th Pro Leu CCG CTG β-226th Val Ile GTC ATC101 α-13th Ile Leu ATC CTC 1.65 times 1.29 times β-40th Thr Ile ACG ATTβ-61st Ala Val GCC GTC β-206th Pro Leu CCG CTG β-226th Val Ile GTC ATC

Reference Example 41 Construction of a Transformant (102) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (102) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ac) amino acidsubstitution sites as shown in Table 39, the plasma described in Example60 of Patent Document 2 was used as the template, and the ribosomebinding sequence was modified according to the method described inExample 1 to prepare a plasmid (102) encoding the above nitrilehydratase variant. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (102).

TABLE 39 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 102 β-51st Phe Val TTC GTCβ-108th Glu Asp GAG GAT

Example 59 Construction of a Transformant (103) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (103) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (s) and (ab) aminoacid substitution sites as shown in Table 40, the plasmid (98) recoveredfrom the transformant (98) described in the above Reference Example 40was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16, 29 and 59 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (103) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (103). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (103) had sequences according to the purpose in whichmutation of 92nd Asp in the α-subunit with Glu, mutation of 4th Val inthe β-subunit with Met and mutation of 206th Pro in the β-subunit withLeu were newly added to the plasmid (98) of Reference Example 40. In theproduction of an amide compound using the thus obtained transformant(103) and the transformant (98) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu, mutation of 4th Val in the β-subunit with Met and mutation of206th Pro in the β-subunit with Leu were newly added to the transformant(103), so that the initial reaction rate was improved by 2.50 times andthermal stability was improved by 1.57 times, as compared to those ofthe transformant (98).

Example 60 Construction of a Transformant (104) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (104) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (s) and (ah) aminoacid substitution sites as shown in Table 40, the plasmid (37) recoveredfrom the transformant (37) described in the above Reference Example 18was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16, 29 and 59 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (104) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (104). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (104) had sequences according to the purpose in whichmutation of 92nd Asp in the α-subunit with Glu, mutation of 4th Val inthe β-subunit with Met and mutation of 206th Pro in the β-subunit withLeu were newly added to the plasmid (37) of Reference Example 18. In theproduction of an amide compound using the thus obtained transformant(104) and the transformant (37) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu, mutation of 4th Val in the β-subunit with Met and mutation of206th Pro in the β-subunit with Leu were newly added to the transformant(104), so that the initial reaction rate was improved by 1.82 times andthermal stability was improved by 1.41 times, as compared to those ofthe transformant (37).

Example 61 Construction of a Transformant (105) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (105) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (s) and (ac) aminoacid substitution sites as shown in Table 40, the plasmid (102)recovered from the transformant (102) described in the above ReferenceExample 41 was used as the template, and the primers having the sequencedefined in SEQ ID Nos: 16, 29 and 59 in the Sequence Listing were usedfor repeatedly carrying out the method described in Reference Example 2per mutation point, whereby a plasmid (105) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (105). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (105) had sequences according to the purpose in whichmutation of 92nd Asp in the α-subunit with Glu, mutation of 4th Val inthe β-subunit with Met and mutation of 206th Pro in the β-subunit withLeu were newly added to the plasmid (102) of Reference Example 41. Inthe production of an amide compound using the thus obtained transformant(105) and the transformant (102) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu, mutation of 4th Val in the β-subunit with Met and mutation of206th Pro in the β-subunit with Leu were newly added to the transformant(105), so that the initial reaction rate was improved by 1.67 times andthermal stability was improved by 1.61 times, as compared to those ofthe transformant (102).

TABLE 40 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-92nd, β-4th α-92nd, β-4th formant Mutated Substitu-Substitu- Substitu- Substitu- and β-206th and β-206th No. Site tion tiontion tion Substitution Substitution 103 α-92nd Asp Glu GAC GAG 2.50times 1.57 times α-148th Gly Asp GGC GAC α-204th Val Arg GTC CGC β-4thVal Met GTG ATG β-206th Pro Leu CCG CTG 104 α-36th Thr Met ACG ATG 1.82times 1.41 times α-92nd Asp Glu GAC GAG α-148th Gly Asp GGC GAC α-204thVal Arg GTC CGC β-4th Val Met GTG ATG β-206th Pro Leu CCG CTG 105 α-92ndAsp Glu GAC GAG 1.67 times 1.61 times β-4th Val Met GTG ATG β-51st PheVal TTC GTC β-108th Glu Asp GAG GAT β-206th Pro Leu CCG CTG

Reference Example 42 Construction of a Transformant (106) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (106) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (az) amino acidsubstitution sites as shown in Table 41, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 65 and 53 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (106) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (106).

TABLE 41 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 106 α-48th Asn Glu AAC GAAα-146th Arg Gly CGG GGG

Example 62 Construction of a Transformant (107) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (107) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (t) and (ac) aminoacid substitution sites as shown in Table 42, the plasmid (102)recovered from the transformant (102) described in the above ReferenceExample 41 was used as the template, and the primers having the sequencedefined in SEQ ID Nos: 24, 37 and 60 in the Sequence Listing were usedfor repeatedly carrying out the method described in Reference Example 2per mutation point, whereby a plasmid (107) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (107). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (107) had sequences according to the purpose in whichmutation of 197th Gly in the α-subunit with Cys, mutation of 107th Proin the β-subunit with Met and mutation of 230th Ala in the β-subunitwith Glu were newly added to the plasmid (102) of Reference Example 41.In the production of an amide compound using the thus obtainedtransformant (107) and the transformant (102) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys, mutation of 107th Pro in the β-subunit with Met and mutationof 230th Ala in the β-subunit with Glu were newly added to thetransformant (107), so that the initial reaction rate was improved by2.11 times and thermal stability was improved by 1.88 times, as comparedto those of the transformant (102).

Example 63 Construction of a Transformant (108) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (108) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (t) and (al) aminoacid substitution sites as shown in Table 42, the plasmid (70) recoveredfrom the transformant (70) described in the above Reference Example 30was used as the template, and the primers having the sequence defined inSEQ ID Nos: 24, 37 and 60 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (108) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (108). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (108) had sequences according to the purpose in whichmutation of 197th Gly in the α-subunit with Cys, mutation of 107th Proin the β-subunit with Met and mutation of 230th Ala in the β-subunitwith Glu were newly added to the plasmid (70) of Reference Example 30.In the production of an amide compound using the thus obtainedtransformant (108) and the transformant (70) to be its base, the initialreaction rate and thermal stability were compared in the same manner asin Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys, mutation of 107th Pro in the β-subunit with Met and mutationof 230th Ala in the β-subunit with Glu were newly added to thetransformant (108), so that the initial reaction rate was improved by1.98 times and thermal stability was improved by 2.34 times, as comparedto those of the transformant (70).

Example 64 Construction (109) of a Transformant (109) Substituted AminoAcid Having Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (109) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (t) and (az) aminoacid substitution sites as shown in Table 42, the plasmid (106)recovered from the transformant (106) described in the above ReferenceExample 42 was used as the template, and the primers having the sequencedefined in SEQ ID Nos: 24, 37 and 60 in the Sequence Listing were usedfor repeatedly carrying out the method described in Reference Example 2per mutation point, whereby a plasmid (109) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (109). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (109) had sequences according to the purpose in whichmutation of 197th Gly in the α-subunit with Cys, mutation of 107th Proin the β-subunit with Met and mutation of 230th Ala in the β-subunitwith Glu were newly added to the plasmid (106) of Reference Example 43.In the production of an amide compound using the thus obtainedtransformant (109) and the transformant (106) to be its base, theinitial reaction rate and thermal stability were compared in the samemanner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys, mutation of 107th Pro in the β-subunit with Met and mutationof 230th Ala in the β-subunit with Glu were newly added to thetransformant (109), so that the initial reaction rate was improved by2.05 times and thermal stability was improved by 1.62 times, as comparedto those of the transformant (106).

TABLE 42 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-197th, β-107th α-197th, β-107th formant MutatedSubstitu- Substitu- Substitu- Substitu- and β-230th and β-230th No. Sitetion tion tion tion Substitution Substitution 107 α-197th Gly Cys GGCTGC 2.11 times 1.88 times β-51st Phe Val TTC GTC β-107th Pro Met CCC ATGβ-108th Glu Asp GAG GAT β-230th Ala Glu GCG GAG 108 α-197th Gly Cys GGCTGC 1.98 times 2.34 times β-41st Phe Ile TTC ATC β-51st Phe Val TTC GTCβ-107th Pro Met CCC ATG β-108th Glu Asp GAG GAT β-230th Ala Glu GCG GAG109 α-48th Asn Glu AAC GAA 2.05 times 1.62 times α-197th Gly Cys GGC TGCβ-107th Pro Met CCC ATG β-146th Arg Gly CGG GGG β-230th Ala Glu GCG GAG

Reference Example 43 Construction of a Transformant (110) SubstitutedAmino Acid Having Nitrile Hydratase Activity

In order to obtain a transformant (110) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (ba) amino acidsubstitution sites as shown in Table 43, introduction of site-specificmutation was performed using the mutagenesis kit described in the aboveReference Example 2. The plasmid (1) expressing nitrile hydratase withthe modified ribosome binding sequence described in Example 1 was usedas the template, and the primers having the sequence defined in SEQ IDNos: 66 and 67 in the Sequence Listing were used for repeatedly carryingout the method described in Reference Example 2 per mutation point,whereby a plasmid (110) encoding the above nitrile hydratase variant wasprepared. A competent cell of Escherichia coli HB101 (manufactured byToyobo Co., Ltd.) was transformed with the plasmid to obtain atransformant (110).

TABLE 43 Change in Amino Change in Trans- Acid Sequence Base Sequenceformant Mutated Before After Before After No. Site SubstitutionSubstitution Substitution Substitution 110 α-36th Thr Trp ACG TGGβ-176th Tyr Cys TAC TGC

Example 65 Construction of a Transformant (111) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (111) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (u) and (aq) aminoacid substitution sites as shown in Table 44, the plasmid (38) recoveredfrom the transformant (38) described in the above Reference Example 19was used as the template, and the primers having the sequence defined inSEQ ID Nos: 33 and 69 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (111) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (111). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (111) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn, mutation of 230th Ala in the β-subunit with Glu andmutation of 231st Ala in the β-subunit with Val were newly added to theplasmid (38) of Reference Example 19. In the production of an amidecompound using the thus obtained transformant (111) and the transformant(38) to be its base, the initial reaction rate and thermal stabilitywere compared in the same manner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn, mutation of 230th Ala in the β-subunit with Glu and mutationof 231st Ala in the β-subunit with Val were newly added to thetransformant (111), so that the initial reaction rate was improved by1.46 times and thermal stability was improved by 1.43 times, as comparedto those of the transformant (38).

Example 66 Construction of a Transformant (112) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (112) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (u) and (ap) aminoacid substitution sites as shown in Table 44, the plasmid (9) recoveredfrom the transformant (9) described in the above Reference Example 5 wasused as the template, and the primers having the sequence defined in SEQID Nos: 33 and 69 in the Sequence Listing were used for repeatedlycarrying out the method described in Reference Example 2 per mutationpoint, whereby a plasmid (112) encoding the above nitrile hydratasevariant was prepared. A competent cell of Escherichia coli HB101(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid toobtain a transformant (112). Moreover, the plasmid was prepared from theabove-mentioned microbial cells by the alkaline SDS extraction method,and the base sequence of the nitrile hydratase gene was determined usinga DNA sequencer. Then, it was confirmed that the transformant (112) hadsequences according to the purpose in which mutation of 79th His in theβ-subunit with Asn, mutation of 230th

Ala in the β-subunit with Glu and mutation of 231st Ala in the β-subunitwith Val were newly added to the plasmid (9) of Reference Example 5. Inthe production of an amide compound using the thus obtained transformant(112) and the transformant (9) to be its base, the initial reaction rateand thermal stability were compared in the same manner as in Example 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn, mutation of 230th Ala in the β-subunit with Glu and mutationof 231st Ala in the β-subunit with Val were newly added to thetransformant (112), so that the initial reaction rate was improved by1.42 times and thermal stability was improved by 1.66 times, as comparedto those of the transformant (9).

Example 67 Construction of a Transformant (113) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (113) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (u) and (ba) aminoacid substitution sites as shown in Table 44, the plasmid (110)recovered from the transformant (110) described in the above ReferenceExample 43 was used as the template, and the primers having the sequencedefined in SEQ ID Nos: 33 and 69 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (113) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (113). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (113) had sequences according to the purpose in whichmutation of 79th His in the β-subunit with Asn, mutation of 230th Ala inthe β-subunit with Glu and mutation of 231st Ala in the β-subunit withVal were newly added to the plasmid (110) of Reference Example 43. Inthe production of an amide compound using the thus obtained transformant(113) and the transformant (110) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 79th His in the β-subunitwith Asn, mutation of 230th Ala in the β-subunit with Glu and mutationof 231st Ala in the β-subunit with Val were newly added to thetransformant (113), so that the initial reaction rate was improved by1.39 times and thermal stability was improved by 1.38 times, as comparedto those of the transformant (110).

TABLE 44 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After β-79th, β-230th β-79th, β-230th formant MutatedSubstitu- Substitu- Substitu- Substitu- and β-231st and β-231st No. Sitetion tion tion tion Substitution Substitution 111 α-6th Leu Thr CTG ACG1.46 times 1.43 times α-19th Ala Val GCG GTG α-126th Phe Tyr TTC TACβ-48th Leu Val CTG GTG β-79th His Asn CAC AAC β-108th Glu Arg GAG CGGβ-212th Ser Tyr TCC TAC β-230th Ala Glu GCG GAG β-231st Ala Val GCC GTC112 α-6th Leu Thr CTG ACG 1.42 times 1.66 times α-19th Ala Val GCG GTGα-126th Phe Tyr TTC TAC β-46th Met Lys ATG AAG β-79th His Asn CAC AACβ-108th Glu Arg GAG CGG β-212th Ser Tyr TCC TAC β-230th Ala Glu GCG GAGβ-231st Ala Val GCC GTC 113 α-36th Thr Trp ACG TGG 1.39 times 1.38 timesβ-79th His Asn CAC AAC β-176th Tyr Cys TAC TGC β-230th Ala Glu GCG GAGβ-231st Ala Val GCC GTC

Example 68 Construction of a Transformant (114) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (114) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (v) and (al) aminoacid substitution sites as shown in Table 45, the plasmid (70) recoveredfrom the transformant (70) described in the above Reference Example 30was used as the template, and the primers having the sequence defined inSEQ ID Nos: 16, 38 and 70 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (114) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (114). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (114) had sequences according to the purpose in whichmutation of 92nd Asp in the α-subunit with Glu, mutation of 24th Val inthe β-subunit with Ile and mutation of 226th Val in the β-subunit withIle were newly added to the plasmid (70) of Reference Example 30. In theproduction of an amide compound using the thus obtained transformant(114) and the transformant (70) to be its base, the initial reactionrate and thermal stability were compared in the same manner as inExample 1.

As a result, it was found that mutation of 92nd Asp in the α-subunitwith Glu, mutation of 24th Val in the β-subunit with Ile and mutation of226th Val in the β-subunit with Ile were newly added to the transformant(114), so that the initial reaction rate was improved by 2.43 times andthermal stability was improved by 1.63 times, as compared to those ofthe transformant (70).

TABLE 45 Improvement of Change in Amino Acid Change in Base Improvementof Thermal Sequence Sequence Reaction Rate by Stability by Trans- BeforeAfter Before After α-92nd, β-24th α-92nd, β-24th formant MutatedSubstitu- Substitu- Substitu- Substitu- and β-226th and β-226th No. Sitetion tion tion tion Substitution Substitution 114 α-92nd Asp Glu GAC GAG2.43 times 1.63 times β-24th Val Ile GTC ATC β-41st Phe Ile TTC ATCβ-51st Phe Val TTC GTC β-108th Glu Asp GAG GAT β-226th Val Ile GTC ATC

Example 69 Construction of a Transformant (115) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (115) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (w) and (al) aminoacid substitution sites as shown in Table 46, the plasmid (70) recoveredfrom the transformant (70) described in the above Reference Example 30was used as the template, and the primers having the sequence defined inSEQ ID Nos: 24, 37, 60 and 70 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (115) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (115). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (115) had sequences according to the purpose in whichmutation of 197th Gly in the α-subunit with Cys, mutation of 24th Val inthe β-subunit with Ile, mutation of 107th Pro in the β-subunit with Met,and mutation of 230th Ala in the β-subunit with Glu were newly added tothe plasmid (70) of Reference Example 30. In the production of an amidecompound using the thus obtained transformant (115) and the transformant(70) to be its base, the initial reaction rate and thermal stabilitywere compared in the same manner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys, mutation of 24th Val in the β-subunit with Ile, mutation of107th Pro in the β-subunit with Met, and mutation of 230th Ala in theβ-subunit with Glu were newly added to the transformant (115), so thatthe initial reaction rate was improved by 2.23 times and thermalstability was improved by 2.51 times, as compared to those of thetransformant (70).

Example 70 Construction of a Transformant (116) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (116) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (w) and (az) aminoacid substitution sites as shown in Table 46, the plasmid (106)recovered from the transformant (106) described in the above ReferenceExample 42 was used as the template, and the primers having the sequencedefined in SEQ ID Nos: 24, 37, 60 and 70 in the Sequence Listing wereused for repeatedly carrying out the method described in ReferenceExample 2 per mutation point, whereby a plasmid (116) encoding the abovenitrile hydratase variant was prepared. A competent cell of Escherichiacoli HB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (116). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (116) had sequences according to the purpose in whichmutation of 197th Gly in the α-subunit with Cys, mutation of 24th Val inthe β-subunit with Ile, mutation of 107th Pro in the β-subunit with Met,and mutation of 230th Ala in the β-subunit with Glu were newly added tothe plasmid (106) of Reference Example 42. In the production of an amidecompound using the thus obtained transformant (116) and the transformant(106) to be its base, the initial reaction rate and thermal stabilitywere compared in the same manner as in Example 1.

As a result, it was found that mutation of 197th Gly in the α-subunitwith Cys, mutation of 24th Val in the β-subunit with Ile, mutation of107th Pro in the β-subunit with Met, and mutation of 230th Ala in theβ-subunit with Glu were newly added to the transformant (116), so thatthe initial reaction rate was improved by 2.35 times and thermalstability was improved by 1.87 times, as compared to those of thetransformant (106).

TABLE 46 Improvement of Improvement of Thermal Change in Amino AcidChange in Base Reaction Rate by Stability by Sequence Sequence α-197th,β-24th, α-197th, β-24th, Trans- Before After Before After β-107th andβ-107th and formant Mutated Substitu- Substitu- Substitu- Substitu-β-230th β-230th No. Site tion tion tion tion Substitution Substitution115 α-197th Gly Cys GGC TGC 2.23 times 2.51 times β-24th Val Ile GTC ATCβ-41st Phe Ile TTC ATC β-51st Phe Val TTC GTC β-107th Pro Met CCC ATGβ-108th Glu Asp GAG GAT β-230th Ala Glu GCG GAG 116 α-48th Asn Glu AACGAA 2.35 times 1.87 times α-197th Gly Cys GGC TGC β-24th Val Ile GTC ATCβ-107th Pro Met CCC ATG β-146th Arg Gly CGG GGG β-230th Ala Glu GCG GAG

Example 71 Construction of a Transformant (117) Substituted Amino AcidHaving Improved Nitrile Hydratase Activity and Improved ThermalStability

In order to obtain a transformant (117) expressing the nitrile hydratasevariant obtained by mutating nitrile hydratase at (x) and (aq) aminoacid substitution sites as shown in Table 47, the plasmid (38) recoveredfrom the transformant (38) described in the above Reference Example 19was used as the template, and the primers having the sequence defined inSEQ ID Nos: 33, 69 and 70 in the Sequence Listing were used forrepeatedly carrying out the method described in Reference Example 2 permutation point, whereby a plasmid (117) encoding the above nitrilehydratase variant was prepared. A competent cell of Escherichia coliHB101 (manufactured by Toyobo Co., Ltd.) was transformed with theplasmid to obtain a transformant (117). Moreover, the plasmid wasprepared from the above-mentioned microbial cells by the alkaline SDSextraction method, and the base sequence of the nitrile hydratase genewas determined using a DNA sequencer. Then, it was confirmed that thetransformant (117) had sequences according to the purpose in whichmutation of 24th Val in the β-subunit with Ile, mutation of 79th His inthe β-subunit with Asn, mutation of 230th Ala in the β-subunit with Glu,and mutation of 231st Ala in the β-subunit with Val were newly added tothe plasmid (38) of Reference Example 19. In the production of an amidecompound using the thus obtained transformant (117) and the transformant(38) to be its base, the initial reaction rate and thermal stabilitywere compared in the same manner as in Example 1.

As a result, it was found that mutation of 24th Val in the β-subunitwith Ile, mutation of 79th His in the β-subunit with Asn, mutation of230th Ala in the β-subunit with Glu, and mutation of 231st Ala in theβ-subunit with Val were newly added to the transformant (117), so thatthe initial reaction rate was improved by 1.73 times and thermalstability was improved by 1.50 times, as compared to those of thetransformant (38).

TABLE 47 Improvement of Improvement of Thermal Change in Amino AcidChange in Base Reaction Rate by Stability by Sequence Sequence β-24th,β-79th, β-24th, β-79th, Trans- Before After Before After β-230th andβ-230th and formant Mutated Substitu- Substitu- Substitu- Substitu-β-231st β-231st No. Site tion tion tion tion Substitution Substitution117 α-6th Leu Thr CTG ACG 1.73 times 1.50 times α-19th Ala Val GCG GTGα-126th Phe Tyr TTC TAC β-24th Val Ile GTC ATC β-48th Leu Val CTG GTGβ-79th His Asn CAC AAC β-108th Glu Arg GAG CGG β-212th Ser Tyr TCC TACβ-230th Ala Glu GCG GAG β-231st Ala Val GCC GTC

1. A nitrile hydratase variant comprising substitution of at least oneamino acid with another amino acid to improve two or more properties ofnitrile hydratase by substitution of 1, 2 or 3 amino acids, wherein saidproperties to be improved are the initial reaction rate and thermalstability, and wherein the nitrile hydratase variant comprises anα-subunit defined in SEQ ID No: 1 in the Sequence Listing and aβ-subunit defined in SEQ ID No: 2 in the Sequence Listing, andsubstitution of at least one amino acid with another amino acid selectedfrom substitution sites of the amino acid consisting of the following(b) to (l): (a) 92nd of α-subunit; (b) 94th of α-subunit; (c) 197th ofα-subunit; (d) 4th of β-subunit; (e) 24th of β-subunit; (f) 79th ofβ-subunit; (g) 96th of β-subunit; (h) 107th of β-subunit; (i) 226th ofβ-subunit; (j) 110th of β-subunit and 231st of β-subunit; (k) 206th ofβ-subunit and 230th of β-subunit; and (l) 13th of α-subunit, 27th ofα-subunit and 110th of β-subunit, wherein Ile is substituted by Leu when13th amino acid of the α-subunit is substituted, Met is substituted byIle when the 27th amino acid of the α-subunit is substituted, Asp issubstituted by Glu when the 92nd amino acid of the α-subunit issubstituted, Met is substituted by Ile when the 94th amino acid of theα-subunit is substituted, Gly is substituted by Cys when the 197th aminoacid of the α-subunit is substituted, Val is substituted by Met when the4th amino acid of the β-subunit is substituted, Val is substituted byIle when the 24th amino acid of the β-subunit is substituted, His issubstituted by Asn when the 79th amino acid of the β-subunit issubstituted, Gln is substituted by Arg when the 96th amino acid of theβ-subunit is substituted, Pro is substituted by Met when the 107th aminoacid of the β-subunit is substituted, Glu is substituted by Asn when the110th amino acid of the β-subunit is substituted, Pro is substituted byLeu when the 206th amino acid of the β-subunit is substituted, Val issubstituted by Ile when the 226th amino acid of the β-subunit issubstituted, Ala is substituted by Glu when the 230th amino acid of theβ-subunit is substituted, and Ala is substituted by Val when the 231stamino acid of the β-subunit is substituted.
 2. The nitrile hydratasevariant according to claim 1, comprising substitution of at least oneamino acid with another amino acid selected from substitution sites ofthe amino acid consisting of the following (m) to (x): (m) in case of(b) or (g), 13th Ile in the α-subunit is substituted by Leu; (n) in caseof (b) or (h), 27th Met in the α-subunit is substituted by Ile; (o) (d)and (f); (p) in case of (f), 230th Ala in the β-subunit is substitutedby Glu; (q) (a) and (i); (r) in case of (i), 13th Ile in the α-subunitis substituted by Leu and 206th Pro in the β-subunit is substituted byLeu; (s) in case of (a) and (d), 206th Pro in the β-subunit issubstituted by Leu; (t) in case of (c) and (h), 230th Ala in theβ-subunit is substituted by Glu; (u) in case of (f), 230th Ala in theβ-subunit is substituted by Glu and 231st Ala in the β-subunit issubstituted by Val; (v) (a) and (e) and (i); (w) in case of (c) and (e)and (h), 230th Ala in the β-subunit is substituted by Glu; and (x) incase of (e) and (f), 230th Ala in the β-subunit is substituted by Gluand 231st Ala in the β-subunit is substituted by Val.
 3. The nitrilehydratase variant according to claim 1, further comprising substitutionof at least one amino acid with another amino acid selected from thegroup consisting of (a), (c), (f), (i), (h), 230th of the β-subunit and231st of the β-subunit in case of (e) is substituted with another aminoacid.
 4. The nitrile hydratase variant according to claim 1, furthercomprising substitution of at least one amino acid selected fromsubstitutions of the amino acid consisting of the following (aa) to(br): (aa) 36th Thr in the α-subunit is substituted by Met and 126th Phein the α-subunit is substituted by Tyr; (ab) 148th Gly in the α-subunitis substituted by Asp and 204th Val in the α-subunit is substituted byArg; (ac) 51st Phe in the β-subunit is substituted by Val and 108th Gluin the β-subunit is substituted by Asp; (ad) 118th Phe in the β-subunitis substituted by Val and 200th Ala in the β-subunit is substituted byGlu; (ae) 160th Arg in the β-subunit is substituted by Trp and 186th Leuin the β-subunit is substituted by Arg; (af) 6th Leu in the α-subunit issubstituted by Thr, 36th Thr in the α-subunit is substituted by Met, and126th Phe in the α-subunit is substituted by Tyr; (ag) 19th Ala in theα-subunit is substituted by Val, 71st Arg in the α-subunit issubstituted by His, and 126th Phe in the α-subunit is substituted byTyr; (ah) 36th Thr in the α-subunit is substituted by Met, 148th Gly inthe α-subunit is substituted by Asp, and 204th Val in the α-subunit issubstituted by Arg; (ai) 10th Thr in the β-subunit is substituted byAsp, 118th Phe in the β-subunit is substituted by Val, and 200th Ala inthe β-subunit is substituted by Glu; (aj) 37th Phe in the β-subunit issubstituted by Leu, 108th Glu in the β-subunit is substituted by Asp,and 200th Ala in the β-subunit is substituted by Glu; (ak) 37th Phe inthe β-subunit is substituted by Val, 108th Glu in the β-subunit issubstituted by Asp, and 200th Ala in the β-subunit is substituted byGlu; (al) 41st Phe in the β-subunit is substituted by Ile, 51st Phe inthe β-subunit is substituted by Val, and 108th Glu in the β-subunit issubstituted by Asp; (am) 46th Met in the β-subunit is substituted byLys, 108th Glu in the β-subunit is substituted by Arg, and 212th Ser inthe β-subunit is substituted by Tyr; (an) 48th Leu in the β-subunit issubstituted by Val, 108th Glu in the β-subunit is substituted by Arg,and 212th Ser in the β-subunit is substituted by Tyr; (ao) 127th Leu inthe β-subunit is substituted by Ser, 160th Arg in the β-subunit issubstituted by Trp, and 186th Leu in the β-subunit is substituted byArg; (ap) 6th Leu in the α-subunit is substituted by Thr, 19th Ala inthe α-subunit is substituted by Val, 126th Phe in the α-subunit issubstituted by Tyr, 46th Met in the β-subunit is substituted by Lys,108th Glu in the β-subunit is substituted by Arg, and 212th Ser in theβ-subunit is substituted by Tyr; (aq) 6th Leu in the α-subunit issubstituted by Thr, 19th Ala in the α-subunit is substituted by Val,126th Phe in the α-subunit is substituted by Tyr, 48th Leu in theβ-subunit is substituted by Val, 108th Glu in the β-subunit issubstituted by Arg, and 212th Ser in the β-subunit is substituted byTyr; (ar) 6th Leu in the α-subunit is substituted by Ala, 19th Ala inthe α-subunit is substituted by Val, 126th Phe in the α-subunit issubstituted by Tyr, 127th Leu in the β-subunit is substituted by Ser,160th Arg in the β-subunit is substituted by Trp, and 186th Leu in theβ-subunit is substituted by Arg; (as) 6th Leu in the α-subunit issubstituted by Thr, 36th Thr in the α-subunit is substituted by Met,126th Phe in the α-subunit is substituted by Tyr, 10th Thr in theβ-subunit is substituted by Asp, 118th Phe in the β-subunit issubstituted by Val, and 200th Ala in the β-subunit is substituted byGlu; (at) 19th Ala in the α-subunit is substituted by Val, 71st Arg inthe α-subunit is substituted by His, 126th Phe in the α-subunit issubstituted by Tyr, 37th Phe in the β-subunit is substituted by Leu,108th Glu in the β-subunit is substituted by Asp, and 200th Ala in theβ-subunit is substituted by Glu; (au) 19th Ala in the α-subunit issubstituted by Val, 71st Arg in the α-subunit is substituted by His,126th Phe in the α-subunit is substituted by Tyr, 37th Phe in theβ-subunit is substituted by Val, 108th Glu in the β-subunit issubstituted by Asp, and 200th Ala in the β-subunit is substituted byGlu; (av) 36th Thr in the α-subunit is substituted by Met, 148th Gly inthe α-subunit is substituted by Asp, 204th Val in the α-subunit issubstituted by Arg, 41st Phe in the β-subunit is substituted by Ile,51st Phe in the β-subunit is substituted by Val, and 108th Glu in theβ-subunit is substituted by Asp; (aw) 148th Gly in the α-subunit issubstituted by Asp, 204th Val in the α-subunit is substituted by Arg,108th Glu in the β-subunit is substituted by Asp, and 200th Ala in theβ-subunit is substituted by Glu; (ax) 36th Thr in the α-subunit issubstituted by Gly and 188th Thr in the α-subunit is substituted by Gly;(ay) 36th Thr in the α-subunit is substituted by Ala and 48th Asn in theα-subunit is substituted by Gln; (az) 48th Asn in the α-subunit issubstituted by Glu and 146th Arg in the β-subunit is substituted by Gly;(ba) 36th Thr in the α-subunit is substituted by Trp and 176th Tyr inthe β-subunit is substituted by Cys; (bb) 176th Tyr in the β-subunit issubstituted by Met and 217th Asp in the β-subunit is substituted by Gly;(bc) 36th Thr in the α-subunit is substituted by Ser, and 33rd Ala inthe β-subunit is substituted by Val; (bd) 176th Tyr in the β-subunit issubstituted by Ala and 217th Asp in the β-subunit is substituted by Val;(be) 40th Thr in the β-subunit is substituted by Val and 218th Cys inthe β-subunit is substituted by Met; (bf) 33rd Ala in the β-subunit issubstituted by Met and 176th Tyr in the β-subunit is substituted by Thr;(bg) 40th Thr in the β-subunit is substituted by Leu and 217th Asp inthe β-subunit is substituted by Leu; (bh) 40th Thr in the β-subunit issubstituted by Ile and 61st Ala in the β-subunit is substituted by Val;(bi) 61st Ala in the β-subunit is substituted by Thr and 218th Cys inthe β-subunit is substituted by Ser; (bj) 112th Lys in the β-subunit issubstituted by Val and 217th Asp in the β-subunit is substituted by Met;(bk) 61st Ala in the β-subunit is substituted by Trp and 217th Asp inthe β-subunit is substituted by His; (bl) 61st Ala in the β-subunit issubstituted by Leu and 112th Lys in the β-subunit is substituted by Ile;(bm) 146th Arg in the β-subunit is substituted by Gly and 217th Asp inthe β-subunit is substituted by Ser; (bn) 171st Lys in the β-subunit issubstituted by Ala and 217th Asp in the β-subunit is substituted by Thr;(bo) 150th Ala in the β-subunit is substituted by Ser and 217th Asp inthe β-subunit is substituted by Cys; (bp) 61st Ala in the β-subunit issubstituted by Gly and 150th Ala in the β-subunit is substituted by Asn;(bq) 61st Ala in the β-subunit is substituted by Ser and 160th Arg inthe β-subunit is substituted by Met; and (br) 160th Arg in the β-subunitis substituted by Cys and 168th Thr in the β-subunit is substituted byGlu.
 5. The nitrile hydratase variant according to claim 1, comprisingsubstitutions at the following substitution sites (I), (II), (III),(IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII) or (XIV)with another amino acids: (I) 36th of α-subunit, 92nd of α-subunit,148th of α-subunit, 204th of α-subunit, 41st of β-subunit, 51st ofβ-subunit and 108th of β-subunit; (II) 6th of α-subunit, 19th ofα-subunit, 94th of α-subunit, 126th of α-subunit, 46th of β-subunit,108th of β-subunit and 212th of β-subunit; (III) 6th of α-subunit, 19thof α-subunit, 126th of α-subunit, 4th of β-subunit, 127th of β-subunit,160th of β-subunit and 186th of β-subunit; (IV) 19th of α-subunit, 71stof α-subunit, 126th of α-subunit, 37th of β-subunit, 79th of β-subunit,108th of β-subunit and 200th of β-subunit; (V) 6th of α-subunit, 19th ofα-subunit, 126th of α-subunit, 48th of β-subunit, 96th of β-subunit,108th of β-subunit and 212th of β-subunit; (VI) 19th of α-subunit, 71stof α-subunit, 126th of α-subunit, 37th of β-subunit, 107th of β-subunit,108th of β-subunit and 200th of β-subunit; (VII) 13th of α-subunit, 19thof α-subunit, 71st of α-subunit, 126th of α-subunit, 37th of β-subunit,96th of β-subunit, 108th of β-subunit and 200th of β-subunit; (VIII) 6thof α-subunit, 27th of α-subunit, 36th of α-subunit, 126th of α-subunit,10th of β-subunit, 107th of β-subunit, 118th of β-subunit and 200th ofβ-subunit; (IX) 6th of α-subunit, 19th of α-subunit, 126th of α-subunit,48th of β-subunit, 79th of β-subunit, 108th of β-subunit, 212th ofβ-subunit and 230th of β-subunit; (X) 6th of α-subunit, 36th ofα-subunit, 126th of α-subunit, 10th of β-subunit, 118th of β-subunit,200th of β-subunit, 206th of β-subunit and 230th of β-subunit; (XI) 36thof α-subunit, 148th of α-subunit, 204th of α-subunit, 41st of β-subunit,51st of β-subunit, 108th of β-subunit, 206th of β-subunit and 230th ofβ-subunit; (XII) 6th of α-subunit, 19th of α-subunit, 126th ofα-subunit, 48th of β-subunit, 79th of β-subunit, 108th of β-subunit,212th of β-subunit, 230th of β-subunit and 231st of β-subunit; (XIII)6th of α-subunit, 19th of α-subunit, 126th of α-subunit, 46th ofβ-subunit, 79th of β-subunit, 108th of β-subunit, 212th of β-subunit,230th of β-subunit and 231st of β-subunit; (XIV) 6th of α-subunit, 19thof α-subunit, 126th of α-subunit, 24th of β-subunit, 48th of β-subunit,79th of β-subunit, 108th of β-subunit, 212th of β-subunit, 230th ofβ-subunit and 231st of β-subunit.
 6. The nitrile hydratase variantaccording to claim 5, wherein: in case of (I), 36th of α-subunit issubstituted with Met, 92nd of α-subunit is substituted with Glu, 148thof α-subunit is substituted with Asp, 204th of α-subunit is substitutedwith Arg, 41st of β-subunit is substituted with Ile, 51st of β-subunitis substituted with Val, and 108th of β-subunit is substituted with Asp;in case of (II), 6th of α-subunit is substituted with Thr, 19th ofα-subunit is substituted with Val, 94th of α-subunit is substituted withIle, 126th of α-subunit is substituted with Tyr, 46th of β-subunit issubstituted with Lys, 108th of β-subunit is substituted with Arg, and212th of β-subunit is substituted with Tyr; in case of (III), 6th ofα-subunit is substituted with Ala, 19th of α-subunit is substituted withVal, 126th of α-subunit is substituted with Tyr, 4th of β-subunit issubstituted with Met, 127th of β-subunit is substituted with Ser, 160thof β-subunit is substituted with Trp, and 186th of β-subunit issubstituted with Arg; in case of (IV), 19th of α-subunit is substitutedwith Val, 71st of α-subunit is substituted with His, 126th of α-subunitis substituted with Tyr, 37th of β-subunit is substituted with Val, 79thof β-subunit is substituted with Asn, 108th of β-subunit is substitutedwith Asp, and 200th of β-subunit is substituted with Glu; in case of(V), 6th of α-subunit is substituted with Thr, 19th of α-subunit issubstituted with Val, 126th of α-subunit is substituted with Tyr, 48thof β-subunit is substituted with Val, 96th of β-subunit is substitutedwith Arg, 108th of β-subunit is substituted with Arg, and 212th ofβ-subunit is substituted with Tyr; in case of (VI), 19th of α-subunit issubstituted with Val, 71st of α-subunit is substituted with His, 126thof α-subunit is substituted with Tyr, 37th of β-subunit is substitutedwith Val, 107th of β-subunit is substituted with Met, 108th of β-subunitis substituted with Asp, and 200th of β-subunit is substituted with Glu;in case of (VII), 13th of α-subunit is substituted with Leu, 19th ofα-subunit is substituted with Val, 71st of α-subunit is substituted withHis, 126th of α-subunit is substituted with Tyr, 37th of β-subunit issubstituted with Leu, 96th of β-subunit is substituted with Arg, 108thof β-subunit is substituted with Asp, and 200th of β-subunit issubstituted with Glu; in case of (VIII), 6th of α-subunit is substitutedwith Thr, 27th of α-subunit is substituted with Ile, 36th of α-subunitis substituted with Met, 126th of α-subunit is substituted with Tyr,10th of β-subunit is substituted with Asp, 107th of β-subunit issubstituted with Met, 118th of β-subunit is substituted with Val, and200th of β-subunit is substituted with Glu; in case of (IX), 6th ofα-subunit is substituted with Thr, 19th of α-subunit is substituted withVal, 126th of α-subunit is substituted with Tyr, 48th of β-subunit issubstituted with Val, 79th of β-subunit is substituted with Asn, 108thof β-subunit is substituted with Arg, 212th of β-subunit is substitutedwith Tyr, and 230th of β-subunit is substituted with Glu; in case of(X), 6th of α-subunit is substituted with Thr, 36th of α-subunit issubstituted with Met, 126th of α-subunit is substituted with Tyr, 10thof β-subunit is substituted with Asp, 118th of β-subunit is substitutedwith Val, 200th of β-subunit is substituted with Glu, 206th of β-subunitis substituted with Leu, and 230th of β-subunit is substituted with Glu;in case of (XI), 36th of α-subunit is substituted with Met, 148th ofα-subunit is substituted with Asp, 204th of α-subunit is substitutedwith Arg, 41st of β-subunit is substituted with Ile, 51st of β-subunitis substituted with Val, 108th of β-subunit is substituted with Asp,206th of β-subunit is substituted with Leu, and 230th of β-subunit issubstituted with Glu; in case of (XII), 6th of α-subunit is substitutedwith Thr, 19th of α-subunit is substituted with Val, 126th of α-subunitis substituted with Tyr, 48th of β-subunit is substituted with Val, 79thof β-subunit is substituted with Asn, 108th of β-subunit is substitutedwith Arg, 212th of β-subunit is substituted with Tyr, 230th of β-subunitis substituted with Glu, and 231st of β-subunit is substituted with Val;in case of (XIII), 6th of α-subunit is substituted with Thr, 19th ofα-subunit is substituted with Val, 126th of α-subunit is substitutedwith Tyr, 46th of β-subunit is substituted with Lys, 79th of β-subunitis substituted with Asn, 108th of β-subunit is substituted with Arg,212th of β-subunit is substituted with Tyr, 230th of β-subunit issubstituted with Glu, and 231st of β-subunit is substituted with Val; incase of (XIV), 6th of α-subunit is substituted with Thr, 19th ofα-subunit is substituted with Val, 126th of α-subunit is substitutedwith Tyr, 24th of β-subunit is substituted with Ile, 48th of β-subunitis substituted with Val, 79th of β-subunit is substituted with Asn,108th of β-subunit is substituted with Arg, 212th of β-subunit issubstituted with Tyr, 230th of β-subunit is substituted with Glu, and231st of β-subunit is substituted with Val.
 7. A gene encoding thenitrile hydratase variant according to claim
 1. 8. A gene encoding anitrile hydratase variant having a gene encoding the α-subunit definedin SEQ ID No: 3 in the Sequence Listing and a gene encoding theβ-subunit defined in SEQ ID No: 4 in the Sequence Listing, comprisingsubstitution of at least one base selected from substitution sites ofthe base consisting of the following (b) to (l): (a) 274th to 276th ofthe base sequence of SEQ ID No: 3; (b) 280th to 282nd of the basesequence of SEQ ID No: 3; (c) 589th to 591st of the base sequence of SEQID No: 3; (d) 10th to 12th of the base sequence of SEQ ID No: 4; (e)70th to 72st of the base sequence of SEQ ID No: 4; (f) 235th to 237th ofthe base sequence of SEQ ID No: 4; (g) 286th to 288th of the basesequence of SEQ ID No: 4; (h) 319th to 321st of the base sequence of SEQID No: 4; (i) 676th to 678th of the base sequence of SEQ ID No: 4; (j)328th to 330th of the base sequence of SEQ ID No: 4 and 691st to 693rdof the base sequence of SEQ ID No: 4; (k) 616th to 618th of the basesequence of SEQ ID No: 4, and 688th to 690th of the base sequence of SEQID No: 4; and (l) 37th to 39th of the base sequence of SEQ ID No: 3,79th to 81st of the base sequence of SEQ ID No: 3, and 328th to 330th ofthe base sequence of SEQ ID No: 4, wherein ATC is substituted by CTCwhen 37th to 39th of the base sequence of SEQ ID No: 3 are substitutedby another base, ATG is substituted by ATC when 79th to 81th of the basesequence of SEQ ID No: 3 are substituted by another base, GAC issubstituted by GAG when 274th to 276th of the base sequence of SEQ IDNo: 3 are substituted by another base, ATG is substituted by ATC when280th to 282th of the base sequence of SEQ ID No: 3 are substituted byanother base, GGC is substituted by TGC when 589th to 591th of the basesequence of SEQ ID No: 3 are substituted by another base, GTG issubstituted by ATG when 10th to 12th of the base sequence of SEQ ID No:4 are substituted by another base, GTC is substituted by ATC when 70thto 72st of the base sequence of SEQ ID No: 4 are substituted by anotherbase, CAC is substituted by AAC when 235th to 237th of the base sequenceof SEQ ID No: 4 are substituted by another base, CAG is substituted byCGT when 286th to 288th of the base sequence of SEQ ID No: 4 aresubstituted by another base, CCC is substituted by ATG when 319th to321st of the base sequence of SEQ ID No: 4 are substituted by anotherbase, GAG is substituted by AAC when 328th to 330th of the base sequenceof SEQ ID No: 4 are substituted by another base, CCG is substituted byCTG when 616th to 618th of the base sequence of SEQ ID No: 4 aresubstituted by another base, GTC is substituted by ATC when 676th to678th of the base sequence of SEQ ID No: 4 are substituted by anotherbase, GCG is substituted by GAG when 688th to 690th of the base sequenceof SEQ ID No: 4 are substituted by another base, and GCC is substitutedby GTC when 691th to 693th of the base sequence of SEQ ID No: 4 aresubstituted by another base.
 9. The gene encoding a nitrile hydratasevariant according to claim 8, further comprising substitution of atleast one base selected from substitution sites of the base consistingof the following (m) to (x): (m) in case of (b) or (g), 37th to 39th ATCof the base sequence of SEQ ID No: 3 are substituted by CTC; (n) in caseof (b) or (h), 79th to 81st ATG of the base sequence of SEQ ID No: 3 aresubstituted by ATC; (o) (d) and (f); (p) in case of (f), 688th to 690thGCG of the base sequence of SEQ ID No: 4 are substituted by GAG; (q) (a)and (i); (r) in case of (i), 37th to 39th ATC of the base sequence ofSEQ ID No: 3 are substituted by CTC and 616th to 618th CCG of the basesequence of SEQ ID No: 4 are substituted by CTG; (s) in case of (a) and(d), 616th to 618th CCG of the base sequence of SEQ ID No: 4 aresubstituted by CTG; (t) in case of (c) and (h), 688th to 690th GCG ofthe base sequence of SEQ ID No: 4 are substituted by GAG; (u) in case of(f), 688th to 690th GCG of the base sequence of SEQ ID No: 4 aresubstituted by GAG and 691st to 693rd GCC of the base sequence of SEQ IDNo: 4 are substituted by GTC; (v) (a) and (e) and (i); (w) in case of(c) and (e) and (h), 688th to 690th GCG of the base sequence of SEQ IDNo: 4 are substituted by GAG; and (x) in case of (e) and (f), 688th to690th GCG of the base sequence of SEQ ID No: 4 are substituted by GAGand 691st to 693rd GCC of the base sequence of SEQ ID No: 4 aresubstituted by GTC.
 10. The gene encoding a nitrile hydratase variantaccording to claim 8, further comprising substitution of at least onebase with another base selected from substitution sites of the baseconsisting of (a), (c), (f), (i), (h), 688th to 690th of the basesequence of SEQ ID No: 4, and 691st to 693rd of the base sequence of SEQID No: 4, in case of (e), are substituted with another base.
 11. Thegene encoding a nitrile hydratase variant according to claim 8,comprising substitution of at least one base selected from substitutionsites of the base consisting of the following (aa) to (br): (aa) 106thto 108th ACG of the base sequence of SEQ ID No: 3 are substituted byATG, and 376th to 378th TTC of the base sequence of SEQ ID No: 3 aresubstituted by TAC; (ab) 442nd to 444th GGC of the base sequence of SEQID No: 3 are substituted by GAC, and 610th to 612th GTC of the basesequence of SEQ ID No: 3 are substituted by CGC; (ac) 151st to 153rd TTCof the base sequence of SEQ ID No: 4 are substituted by GTC, and 322ndto 324th GAG of the base sequence of SEQ ID No: 4 are substituted byGAT; (ad) 352nd to 354th TTC of the base sequence of SEQ ID No: 4 aresubstituted by GTC, and 598th to 600th GCC of the base sequence of SEQID No: 4 are substituted by GAG; (ae) 478th to 480th CGG of the basesequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTGof the base sequence of SEQ ID No: 4 are substituted by CGG; (af) 16thto 18th CTG of the base sequence of SEQ ID No: 3 are substituted by ACG,106th to 108th ACG of the base sequence of SEQ ID No: 3 are substitutedby ATG, and 376th to 378th TTC of the base sequence of SEQ ID No: 3 aresubstituted by TAC; (ag) 55th to 57th GCG of the base sequence of SEQ IDNo: 3 are substituted by GTG, 211th to 213th CGT of the base sequence ofSEQ ID No: 3 are substituted by CAT, and 376th to 378th TTC of the basesequence of SEQ ID No: 3 are substituted by TAC; (ah) 106th to 108th ACGof the base sequence of SEQ ID No: 3 are substituted by ATG, 442nd to444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC,and 610th to 612th GTC of the base sequence of SEQ ID No: 3 aresubstituted by CGC; (ai) 28th to 30th ACC of the base sequence of SEQ IDNo: 4 are substituted by GAC, 352nd to 354th TTC of the base sequence ofSEQ ID No: 4 are substituted by GTC, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG; (aj) 109th to 111th TTCof the base sequence of SEQ ID No: 4 are substituted by CTC, 322nd to324th GAG of the base sequence of SEQ ID No: 4 are substituted by GAT,and 598th to 600th GCC of the base sequence of SEQ ID No: 4 aresubstituted by GAG; (ak) 109th to 111th TTC of the base sequence of SEQID No: 4 are substituted by GTC, 322nd to 324th GAG of the base sequenceof SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of thebase sequence of SEQ ID No: 4 are substituted by GAG; (al) 121st to123rd TTC of the base sequence of SEQ ID No: 4 are substituted by ATC,151st to 153rd TTC of the base sequence of SEQ ID No: 4 are substitutedby GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4 aresubstituted by GAT; (am) 136th to 138th ATG of the base sequence of SEQID No: 4 are substituted by AAG, 322nd to 324th GAG of the base sequenceof SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of thebase sequence of SEQ ID No: 4 are substituted by TAC; (an) 142nd to144th CTG of the base sequence of SEQ ID No: 4 are substituted by GTG,322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substitutedby CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4 aresubstituted by TAC; (ao) 379th to 381st CTG of the base sequence of SEQID No: 4 are substituted by TCG, 478th to 480th CGG of the base sequenceof SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTG of thebase sequence of SEQ ID No: 4 are substituted by CGG; (ap) 16th to 18thCTG of the base sequence of SEQ ID No: 3 are substituted by ACG, 55th to57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 136th to 138th ATG of the base sequence of SEQ ID No: 4 aresubstituted by AAG, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by CGG, and 634th to 636th TCC of the basesequence of SEQ ID No: 4 are substituted by TAC; (aq) 16th to 18th CTGof the base sequence of SEQ ID No: 3 are substituted by ACG, 55th to57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 142nd to 144th CTG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by CGG, and 634th to 636th TCC of the basesequence of SEQ ID No: 4 are substituted by TAC; (ar) 16th to 18th CTGof the base sequence of SEQ ID No: 3 are substituted by GCG, 55th to57th GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 379th to 381st CTG of the base sequence of SEQ ID No: 4 aresubstituted by TCG, 478th to 480th CGG of the base sequence of SEQ IDNo: 4 are substituted by TGG, and 556th to 558th CTG of the basesequence of SEQ ID No: 4 are substituted by CGG; (as) 16th to 18th CTGof the base sequence of SEQ ID No: 3 are substituted by ACG, 106th to108th ACG of the base sequence of SEQ ID No: 3 are substituted by ATG,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 28th to 30th ACC of the base sequence of SEQ ID No: 4 aresubstituted by GAC, 352nd to 354th TTC of the base sequence of SEQ IDNo: 4 are substituted by GTC, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG; (at) 55th to 57th GCGof the base sequence of SEQ ID No: 3 are substituted by GTG, 211th to213th CGT of the base sequence of SEQ ID No: 3 are substituted by CAT,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 109th to 111th TTC of the base sequence of SEQ ID No: 4 aresubstituted by CTC, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by GAT, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG; (au) 55th to 57th GCGof the base sequence of SEQ ID No: 3 are substituted by GTG, 211th to213th CGT of the base sequence of SEQ ID No: 3 are substituted by CAT,376th to 378th TTC of the base sequence of SEQ ID No: 3 are substitutedby TAC, 109th to 111th TTC of the base sequence of SEQ ID No: 4 aresubstituted by GTC, 322nd to 324th GAG of the base sequence of SEQ IDNo: 4 are substituted by GAT, and 598th to 600th GCC of the basesequence of SEQ ID No: 4 are substituted by GAG; (av) 106th to 108th ACGof the base sequence of SEQ ID No: 3 are substituted by ATG, 442nd to444th GGC of the base sequence of SEQ ID No: 3 are substituted by GAC,610th to 612th GTC of the base sequence of SEQ ID No: 3 are substitutedby CGC, 121st to 123rd TTC of the base sequence of SEQ ID No: 4 aresubstituted by ATC, 151st to 153rd TTC of the base sequence of SEQ IDNo: 4 are substituted by GTC, and 322nd to 324th GAG of the basesequence of SEQ ID No: 4 are substituted by GAT; (aw) 442nd to 444th GGCof the base sequence of SEQ ID No: 3 are substituted by GAC, 610th to612th GTC of the base sequence of SEQ ID No: 3 are substituted by CGC,322nd to 324th GAG of the base sequence of SEQ ID No: 4 are substitutedby GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4 aresubstituted by GAG; (ax) 106th to 108th ACG of the base sequence of SEQID No: 3 are substituted by GGG, and 562nd to 564th ACC of the basesequence of SEQ ID No: 3 are substituted by GGC; (ay) 106th to 108th ACGof the base sequence of SEQ ID No: 3 are substituted by GCG, and 142ndto 144th AAC of the base sequence of SEQ ID No: 3 are substituted byCAA; (az) 142nd to 144th AAC of the base sequence of SEQ ID No: 3 aresubstituted by GAA, and 436th to 438th CGG of the base sequence of SEQID No: 4 are substituted by GGG; (ba) 106th to 108th ACG of the basesequence of SEQ ID No: 3 are substituted by TGG, and 526th to 528th TACof the base sequence of SEQ ID No: 4 are substituted by TGC; (bb) 526thto 528th TAC of the base sequence of SEQ ID No: 4 are substituted byATG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 aresubstituted by GGC; (bc) 106th to 108th ACG of the base sequence of SEQID No: 3 are substituted by TCG, and 97th to 99th GCG of the basesequence of SEQ ID No: 4 are substituted by GTG; (bd) 526th to 528th TACof the base sequence of SEQ ID No: 4 are substituted by GCC, and 649thto 651st GAC of the base sequence of SEQ ID No: 4 are substituted byGTC; (be) 118th to 120th ACG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, and 652nd to 654th TGC of the base sequence of SEQID No: 4 are substituted by ATG; (bf) 97th to 99th GCG of the basesequence of SEQ ID No: 4 are substituted by ATG, and 526th to 528th TACof the base sequence of SEQ ID No: 4 are substituted by ACC; (bg) 118thto 120th ACG of the base sequence of SEQ ID No: 4 are substituted byCTG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4 aresubstituted by CTC; (bh) 118th to 120th ACG of the base sequence of SEQID No: 4 are substituted by ATT, and 181st to 183rd GCC of the basesequence of SEQ ID No: 4 are substituted by GTC; (bi) 181st to 183rd GCCof the base sequence of SEQ ID No: 4 are substituted by ACG, and 652ndto 654th TGC of the base sequence of SEQ ID No: 4 are substituted byTCC; (bj) 334th to 336th AAG of the base sequence of SEQ ID No: 4 aresubstituted by GTG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by ATG; (bk) 181st to 183rd GCC of the basesequence of SEQ ID No: 4 are substituted by TGG, and 649th to 651st GACof the base sequence of SEQ ID No: 4 are substituted by CAC; (bl) 181stto 183rd GCC of the base sequence of SEQ ID No: 4 are substituted byCTC, and 334th to 336th AAG of the base sequence of SEQ ID No: 4 aresubstituted by ATT; (bm) 436th to 438th CGG of the base sequence of SEQID No: 4 are substituted by GGG, and 649th to 651st GAC of the basesequence of SEQ ID No: 4 are substituted by AGC; (bn) 511th to 513th AAGof the base sequence of SEQ ID No: 4 are substituted by GCG, and 649thto 651st GAC of the base sequence of SEQ ID No: 4 are substituted byACC; (bo) 448th to 450th GCG of the base sequence of SEQ ID No: 4 aresubstituted by TCG, and 649th to 651st GAC of the base sequence of SEQID No: 4 are substituted by TGT; (bp) 181st to 183rd GCC of the basesequence of SEQ ID No: 4 are substituted by GGC, and 448th to 450th GCGof the base sequence of SEQ ID No: 4 are substituted by AAT; (bq) 181stto 183rd GCC of the base sequence of SEQ ID No: 4 are substituted byTCG, and 478th to 480th CGG of the base sequence of SEQ ID No: 4 aresubstituted by ATG; and (br) 478th to 480th CGG of the base sequence ofSEQ ID No: 4 are substituted by TGT, and 502nd to 504th ACG of the basesequence of SEQ ID No: 4 are substituted by GAG.
 12. The gene encoding anitrile hydratase variant according to claim 8, comprising substitutionsof the following (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX),(X), (XI), (XII), (XIII) or (XIV): (I) the base sequences of 106th to108th, 274th to 276th, 442nd to 444th and 610th to 612th in SEQ ID No:3, and the base sequences of 121st to 123rd, 151st to 153rd, and 322ndto 324th in SEQ ID No: 4; (II) the base sequences of 16th to 18th, 55thto 57th, 280th to 282nd and 376th to 378th in SEQ ID No: 3, and the basesequences of 136th to 138th, 322nd to 324th and 634th to 636th in SEQ IDNo: 4; (III) the base sequences of 16th to 18th, 55th to 57th, and 376thto 378th in SEQ ID No: 3, and the base sequences of 10th to 12th, 379thto 381st, 478th to 480th and 556th to 558th in SEQ ID No: 4; (IV) thebase sequences of 55th to 57th, 211th to 213th and 376th to 378th in SEQID No: 3, and the base sequences of 109th to 111th, 235th to 237th,322nd to 324th and 598th to 600th in SEQ ID No: 4; (V) the basesequences of 16th to 18th, 55th to 57th, and 376th to 378th in SEQ IDNo: 3, and the base sequences of 142nd to 144th, 286th to 288th, 322ndto 324th and 634th to 636th in SEQ ID No: 4; (VI) the base sequences of55th to 57th, 211th to 213th, and 376th to 378th in SEQ ID No: 3, andthe base sequences of 109th to 111th, 319th to 321st, 322nd to 324th and598th to 600th in SEQ ID No: 4; (VII) the base sequences of 37th to39th, 55th to 57th, 211th to 213th and 376th to 378th in SEQ ID No: 3,and the base sequences of 109th to 111th, 286th to 288th, 322nd to 324thand 598th to 600th in SEQ ID No: 4; (VIII) the base sequences of 16th to18th, 79th to 81st, 106th to 108th and 376th to 378th in SEQ ID No: 3,and the base sequences of 28th to 30th, 319th to 321st, 352nd to 354thand 598th to 600th in SEQ ID No: 4; (IX) the base sequences of 16th to18th, 55th to 57th and 376th to 378th in SEQ ID No: 3, and the basesequences of 142nd to 144th, 235th to 237th, 322nd to 324th, 634th to636th and 688th to 690th in SEQ ID No: 4; (X) the base sequences of 16thto 18th, 106th to 108th and 376th to 378th in SEQ ID No: 3, and the basesequences of 28th to 30th, 352nd to 354th, 598th to 600th, 616th to618th and 688th to 690th in SEQ ID No: 4; (XI) the base sequences of106th to 108th, 422nd to 444th and 610th to 612th in SEQ ID No: 3, andthe base sequences of 121st to 123rd, 151st to 153rd, 322nd to 324th,616th to 618th and 688th to 690th in SEQ ID No: 4; (XII) the basesequences of 16th to 18th, 55th to 57th and 376th to 378th in SEQ ID No:3, and the base sequences of 142nd to 144th, 235th to 237th, 322nd to324th, 634th to 636th, 688th to 690th and 691st to 693rd in SEQ ID No:4; (XIII) the base sequences of 16th to 18th, 55th to 57th and 376th to378th in SEQ ID No: 3, and the base sequences of 136th to 138th, 235thto 237th, 322nd to 324th, 634th to 636th, 688th to 690th and 691st to693rd in SEQ ID No: 4; (XIV) the base sequences of 16th to 18th, 55th to57th and 376th to 378th in SEQ ID No: 3, and the base sequences of 70thto 72st, 142nd to 148th, 235th to 237th, 322nd to 324th, 634th to 636th,688th to 690th and 691st to 693rd in SEQ ID No: 4;
 13. The gene encodinga nitrile hydratase variant according to claim 12, wherein in case ofsubstitutions of (I): 106th to 108th of the base sequence of SEQ ID No:3 are substituted by ATG; 274th to 276th of the base sequence of SEQ IDNo: 3 are substituted by GAG; 442nd to 444th of the base sequence of SEQID No: 3 are substituted by GAC; 610th to 612th of the base sequence ofSEQ ID No: 3 are substituted by CGC; 121st to 123rd of the base sequenceof SEQ ID No: 4 are substituted by ATC; 151st to 153rd of the basesequence of SEQ ID No: 4 are substituted by GTC; and 322nd to 324th ofthe base sequence of SEQ ID No: 4 are substituted by GAT; wherein incase of substitutions of (II): 16th to 18th of the base sequence of SEQID No: 3 are substituted by ACG; 55th to 57th of the base sequence ofSEQ ID No: 3 are substituted by GTG; 280th to 282nd of the base sequenceof SEQ ID No: 3 are substituted by ATC; 376th to 378th of the basesequence of SEQ ID No: 3 are substituted by TAC; 136th to 138th of thebase sequence of SEQ ID No: 4 are substituted by AAG; 322nd to 324th ofthe base sequence of SEQ ID No: 4 are substituted by CGG; and 634th to636th of the base sequence of SEQ ID No: 4 are substituted by TAC;wherein in case of substitutions of (III): 16th to 18th of the basesequence of SEQ ID No: 3 are substituted by GCG; 55th to 57th of thebase sequence of SEQ ID No: 3 are substituted by GTG; 376th to 378th ofthe base sequence of SEQ ID No: 3 are substituted by TAC; 10th to 12thof the base sequence of SEQ ID No: 4 are substituted by ATG; 379th to381st of the base sequence of SEQ ID No: 4 are substituted by TCG; 478thto 480th of the base sequence of SEQ ID No: 4 are substituted by TGG;and 556th to 558th of the base sequence of SEQ ID No: 4 are substitutedby CGG; wherein in case of substitutions of (IV): 55th to 57th of thebase sequence of SEQ ID No: 3 are substituted by GTG; 211th to 213th ofthe base sequence of SEQ ID No: 3 are substituted by CAT; 376th to 378thof the base sequence of SEQ ID No: 3 are substituted by TAC; 109th to111th of the base sequence of SEQ ID No: 4 are substituted by GTC; 235thto 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;322nd to 324th of the base sequence of SEQ ID No: 4 are substituted byGAT; and 598th to 600th of the base sequence of SEQ ID No: 4 aresubstituted by GAG; wherein in case of substitutions of (V): 16th to18th of the base sequence of SEQ ID No: 3 are substituted by ACG; 55thto 57th of the base sequence of SEQ ID No: 3 are substituted by GTG;376th to 378th of the base sequence of SEQ ID No: 3 are substituted byTAC; 142nd to 144th of the base sequence of SEQ ID No: 4 are substitutedby GTG; 286th to 288th of the base sequence of SEQ ID No: 4 aresubstituted by CGT; 322nd to 324th of the base sequence of SEQ ID No: 4are substituted by CGG; and 634th to 636th of the base sequence of SEQID No: 4 are substituted by TAC; wherein in case of substitutions of(VI): 55th to 57th of the base sequence of SEQ ID No: 3 are substitutedby GTG; 211th to 213th of the base sequence of SEQ ID No: 3 aresubstituted by CAT; 376th to 378th of the base sequence of SEQ ID No: 3are substituted by TAC; 109th to 111th of the base sequence of SEQ IDNo: 4 are substituted by GTC; 319th to 321st of the base sequence of SEQID No: 4 are substituted by ATG; 322nd to 324th of the base sequence ofSEQ ID No: 4 are substituted by GAT; and 598th to 600th of the basesequence of SEQ ID No: 4 are substituted by GAG; wherein in case ofsubstitutions of (VII): 37th to 39th of the base sequence of SEQ ID No:3 are substituted by CTC; 55th to 57th of the base sequence of SEQ IDNo: 3 are substituted by GTG; 211th to 213th of the base sequence of SEQID No: 3 are substituted by CAT; 376th to 378th of the base sequence ofSEQ ID No: 3 are substituted by TAC; 109th to 111th of the base sequenceof SEQ ID No: 4 are substituted by CTC; 286th to 288th of the basesequence of SEQ ID No: 4 are substituted by CGT; 322th to 324th of thebase sequence of SEQ ID No: 4 are substituted by GAT; and 598th to 600thof the base sequence of SEQ ID No: 4 are substituted by GAG; wherein incase of substitutions of (VIII): 16th to 18th of the base sequence ofSEQ ID No: 3 are substituted by ACG; 79th to 81st of the base sequenceof SEQ ID No: 3 are substituted by ATC; 106th to 108th of the basesequence of SEQ ID No: 3 are substituted by ATG; 376th to 378th of thebase sequence of SEQ ID No: 3 are substituted by TAC; 28th to 30th ofthe base sequence of SEQ ID No: 4 are substituted by GAC; 319th to 321stof the base sequence of SEQ ID No: 4 are substituted by ATG; 352nd to354th of the base sequence of SEQ ID No: 4 are substituted by GTC; and598th to 600th of the base sequence of SEQ ID No: 4 are substituted byGAG; wherein in case of substitutions of (IX): 16th to 18th of the basesequence of SEQ ID No: 3 are substituted by ACG; 55th to 57th of thebase sequence of SEQ ID No: 3 are substituted by GTG; 376th to 378th ofthe base sequence of SEQ ID No: 3 are substituted by TAC; 142nd to 144thof the base sequence of SEQ ID No: 4 are substituted by GTG; 235th to237th of the base sequence of SEQ ID No: 4 are substituted by AAC; 322ndto 324th of the base sequence of SEQ ID No: 4 are substituted by CGG;634th to 636th of the base sequence of SEQ ID No: 4 are substituted byTAC; and 688th to 690th of the base sequence of SEQ ID No: 4 aresubstituted by GAG; wherein in case of substitutions of (X): 16th to18th of the base sequence of SEQ ID No: 3 are substituted by ACG; 106thto 108th of the base sequence of SEQ ID No: 3 are substituted by ATG;376th to 378th of the base sequence of SEQ ID No: 3 are substituted byTAC; 28th to 30th of the base sequence of SEQ ID No: 4 are substitutedby GAC; 352nd to 354th of the base sequence of SEQ ID No: 4 aresubstituted by GTC; 598th to 600th of the base sequence of SEQ ID No: 4are substituted by GAG; 616th to 618th of the base sequence of SEQ IDNo: 4 are substituted by CTG; and 688th to 690th of the base sequence ofSEQ ID No: 4 are substituted by GAG; wherein in case of substitutions of(XI): 106th to 108th of the base sequence of SEQ ID No: 3 aresubstituted by ATG; 442nd to 444th of the base sequence of SEQ ID No: 3are substituted by GAC; 610th to 612th of the base sequence of SEQ IDNo: 3 are substituted by CGC; 121st to 123rd of the base sequence of SEQID No: 4 are substituted by ATC; 151st to 153rd of the base sequence ofSEQ ID No: 4 are substituted by GTC; 322nd to 324th of the base sequenceof SEQ ID No: 4 are substituted by GAT; 616th to 618th of the basesequence of SEQ ID No: 4 are substituted by CTG; and 688th to 690th ofthe base sequence of SEQ ID No: 4 are substituted by GAG; wherein incase of substitutions of (XII): 16th to 18th of the base sequence of SEQID No: 3 are substituted by ACG; 55th to 57th of the base sequence ofSEQ ID No: 3 are substituted by GTG; 376th to 378th of the base sequenceof SEQ ID No: 3 are substituted by TAC; 142nd to 144th of the basesequence of SEQ ID No: 4 are substituted by GTG; 235th to 237th of thebase sequence of SEQ ID No: 4 are substituted by AAC; 322nd to 324th ofthe base sequence of SEQ ID No: 4 are substituted by CGG; 634th to 636thof the base sequence of SEQ ID No: 4 are substituted by TAC; 688th to690th of the base sequence of SEQ ID No: 4 are substituted by GAG; and691st to 693rd of the base sequence of SEQ ID No: 4 are substituted byGTC; wherein in case of substitutions of (XIII): 16th to 18th of thebase sequence of SEQ ID No: 3 are substituted by ACG; 55th to 57th ofthe base sequence of SEQ ID No: 3 are substituted by GTG; 376th to 378thof the base sequence of SEQ ID No: 3 are substituted by TAC; 136th to138th of the base sequence of SEQ ID No: 4 are substituted by AAG; 235thto 237th of the base sequence of SEQ ID No: 4 are substituted by AAC;322nd to 324th of the base sequence of SEQ ID No: 4 are substituted byCGG; 634th to 636th of the base sequence of SEQ ID No: 4 are substitutedby TAC; 688th to 690th of the base sequence of SEQ ID No: 4 aresubstituted by GAG; and 691st to 693rd of the base sequence of SEQ IDNo: 4 are substituted by GTC; wherein in case of substitutions of (XIV):16th to 18th of the base sequence of SEQ ID No: 3 are substituted byACG; 55th to 57th of the base sequence of SEQ ID No: 3 are substitutedby GTG; 376th to 378th of the base sequence of SEQ ID No: 3 aresubstituted by TAC; 70th to 72st of the base sequence of SEQ ID No: 4are substituted by ATC; 142nd to 148th of the base sequence of SEQ IDNo: 4 are substituted by GTG; 235th to 237th of the base sequence of SEQID No: 4 are substituted by AAC; 322nd to 324th of the base sequence ofSEQ ID No: 4 are substituted by CGG; 634th to 636th of the base sequenceof SEQ ID No: 4 are substituted by TAC; 688th to 690th of the basesequence of SEQ ID No: 4 are substituted by GAG; and 691st to 693rd ofthe base sequence of SEQ ID No: 4 are substituted by GTC.
 14. A linkedDNA comprising further DNA containing a promoter sequence necessary forthe expression of the gene in the upstream region of the 5′-terminal ofthe gene encoding a nitrile hydratase variant according to claim 8, anda ribosome binding sequence contained in SEQ ID No: 7 in the downstreamregion of the 3′-terminal of the promoter.
 15. A plasmid comprising theDNA according to claim
 14. 16. A transformant obtained by transformationof a host cell using the plasmid according to claim
 15. 17. A method forproducing a nitrile hydratase variant, comprising cultivating thetransformant according to claim 16 in a culture medium and producing anitrile hydratase variant based on the nitrile hydratase gene carried bythe plasmid in the transformant.